Methods for treating viral infection using il-28 and il-29 cysteine mutants

ABSTRACT

IL-28A, IL-28B, IL-29, and certain mutants thereof have been shown to have antiviral activity on a spectrum of viral species. Of particular interest is the antiviral activity demonstrated on viruses that infect liver, such as hepatitis B virus and hepatitis C virus. In addition, IL-28A, IL-28B, IL-29, and mutants thereof do not exhibit some of the antiproliferative activity on hematopoietic cells that is observed with interferon treatment. Without the immunosuppressive effects accompanying interferon treatment, IL-28A, IL-28B, and IL-29 will be useful in treating immunocompromised patients for viral infections.

REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 11/858,699, filed Sep. 20, 2007, which is a continuation of U.S.patent application Ser. No. 11/098,662, filed Apr. 4, 2005, which claimsthe benefit of U.S. Patent Application Ser. Nos. 60/559,081, filed Apr.2, 2004, 60/609,238, filed Sep. 13, 2004, and 60/634,144, filed Dec. 8,2004, all of which are herein incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

Strategies for treating infectious disease often focus on ways toenhance immunity. For instance, the most common method for treatingviral infection involves prophylactic vaccines that induce immune-basedmemory responses. Another method for treating viral infection includespassive immunization via immunoglobulin therapy (Meissner, J. Pediatr.124:S17-21, 1994). Administration of Interferon alpha (IFN-α) is anothermethod for treating viral infections such as genital warts (Reichman etal., Ann. Intern. Med. 108:675-9, 1988) and chronic viral infectionslike hepatitis C virus (HCV) (Davis et al., New Engl. J. Med.339:1493-9, 1998) and hepatitis B virus (HBV). For instance, IFN-α andIFN-β are critical for inhibiting virus replication (reviewed by Vilceket al., (Eds.), Interferons and other cytokines, In Fields FundamentalVirology., 3^(rd) ed., Lippincott-Raven Publishers Philadelphia, Pa.,1996, pages 341-365). In response to viral infection, CD4+ T cellsbecome activated and initiate a T-helper type I (TH1) response and thesubsequent cascade required for cell-mediated immunity. That is,following their expansion by specific growth factors like the cytokineIL-2, T-helper cells stimulate antigen-specific CD8+ T-cells,macrophages, and NK cells to kill virally infected host cells. Althoughoftentimes efficacious, these methods have limitations in clinical use.For instance, many viral infections are not amenable to vaccinedevelopment, nor are they treatable with antibodies alone. In addition,IFN's are not extremely effective and they can cause significanttoxicities; thus, there is a need for improved therapies.

Not all viruses and viral diseases are treated identically becausefactors, such as whether an infection is acute or chronic and thepatient's underlying health, influence the type of treatment that isrecommended. Generally, treatment of acute infections in immunocompetentpatients should reduce the disease's severity, decrease complications,and decrease the rate of transmission. Safety, cost, and convenience areessential considerations in recommending an acute antiviral agent.Treatments for chronic infections should prevent viral damage to organssuch as liver, lungs, heart, central nervous system, andgastrointestinal system, making efficacy the primary consideration.

Chronic hepatitis is one of the most common and severe viral infectionsof humans worldwide belonging to the Hepadnaviridae family of viruses.Infected individuals are at high risk for developing liver cirrhosis,and eventually, hepatic cancer. Chronic hepatitis is characterized as aninflammatory liver disease continuing for at least six months withoutimprovement. The majority of patients suffering from chronic hepatitisare infected with either chronic HBV, HCV or are suffering fromautoimmune disease. The prevalence of HCV infection in the generalpopulation exceeds 1% in the United States, Japan, China and SoutheastAsia.

Chronic HCV can progress to cirrhosis and extensive necrosis of theliver. Although chronic HCV is often associated with deposition of typeI collagen leading to hepatic fibrosis, the mechanisms of fibrogenesisremain unknown. Liver (hepatic) fibrosis occurs as a part of thewound-healing response to chronic liver injury. Fibrosis occurs as acomplication of haemochromatosis, Wilson's disease, alcoholism,schistosomiasis, viral hepatitis, bile duct obstruction, toxin exposure,and metabolic disorders. This formation of scar tissue is believed torepresent an attempt by the body to encapsulate the injured tissue.Liver fibrosis is characterized by the accumulation of extracellularmatrix that can be distinguished qualitatively from that in normalliver. Left unchecked, hepatic fibrosis progresses to cirrhosis (definedby the presence of encapsulated nodules), liver failure, and death.

There are few effective treatments for hepatitis. For example, treatmentof autoimmune chronic hepatitis is generally limited toimmunosuppressive treatment with corticosteroids. For the treatment ofHBV and HCV, the FDA has approved administration of recombinant IFN-α.However, IFN-α is associated with a number of dose-dependent adverseeffects, including thrombocytopenia, leukopenia, bacterial infections,and influenza-like symptoms. Other agents used to treat chronic HBV orHCV include the nucleoside analog RIBAVIRIN™ and ursodeoxycholic acid;however, neither has been shown to be very effective. RIBAVIRIN™+IFNcombination therapy for results in 47% rate of sustained viral clearance(Lanford, R. E. and Bigger, C. Virology 293: 1-9 (2002). (See Medicine,(D. C. Dale and D. D. Federman, eds.) (Scientific American, Inc., NewYork), 4:VIII:1-8 (1995)).

Respiratory syncytial virus is the major cause of pneumonia andbronchiolitis in infancy. RSV infects more than half of infants duringtheir first year of exposure, and nearly all are infected after a secondyear. During seasonal epidemics most infants, children, and adults areat risk for infection or reinfection. Other groups at risk for seriousRSV infections include premature infants, immune compromised childrenand adults, and the elderly. Symptoms of RSV infection range from a mildcold to severe bronchiolitis and pneumonia. Respiratory syncytial virushas also been associated with acute otitis media and RSV can berecovered from middle ear fluid. Herpes simplex virus-1 (HSV-1) andherpes simplex virus-2 (HSV-2) may be either lytic or latent, and arethe causative agents in cold sores (HSV-1) and genital herpes, typicallyassociated with lesions in the region of the eyes, mouth, and genitals(HSV-2). These viruses are a few examples of the many viruses thatinfect humans for which there are few adequate treatments available onceinfection has occurred.

The demonstrated activities of the IL-28 and IL-29 cytokine familyprovide methods for treating specific virual infections, for example,liver specific viral infections. The activity of IL-28 and IL-29 alsodemonstrate that these cytokines provide methods for treatingimmunocompromised patients. The methods for these and other uses shouldbe apparent to those skilled in the art from the teachings herein.

DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “level” when referring to immune cells, such as NK cells, Tcells, in particular cytotoxic T cells, B cells and the like, anincreased level is either increased number of cells or enhanced activityof cell function.

The term “level” when referring to viral infections refers to a changein the level of viral infection and includes, but is not limited to, achange in the level of CTLs or NK cells (as described above), a decreasein viral load, an increase antiviral antibody titer, decrease inserological levels of alanine aminotransferase, or improvement asdetermined by histological examination of a target tissue or organ.Determination of whether these changes in level are significantdifferences or changes is well within the skill of one in the art.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

“zcyto20”, “zcyto21”, “zcyto22” are the previous designations for humanIL-28A, IL-29, and IL-28B, respectively and are used interchangeablyherein. IL-28A polypeptides of the present invention are shown in SEQ IDNOs:2, 18, 24, 26, 28, 30, and 36, which are encoded by polynucleotidesequences as shown in SEQ ID NOs:1, 17, 23, 25, 27, 29, and 35,respectively. IL-28B polypeptides of the present invention are shown inSEQ ID NOs:6, 22, 40, 86, 88, 90, 92, 94, 96, 98, and 100, which areencoded by polynucleotide sequences as shown in SEQ ID NOs:5, 21, 39,85, 87, 89, 91, 93, 95, 97, and 99, respectively. IL-29 polypeptides ofthe present invention are shown in SEQ ID NOs:4, 20, 32, 34, 38, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,and 136, which are encoded by polynucleotide sequences as shown in SEQID NOs:3, 19, 31, 33, 37, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, and 135, respectively.

“zcyto24” and “zcyto25” are the previous designations for mouse IL-28Aand IL-28B, and are shown in SEQ ID NOs:7, 8, 9, 10, respectively. Thepolynucleotide and polypeptides are fully described in PCT applicationWO 02/086087 commonly assigned to ZymoGenetics, Inc., incorporatedherein by reference.

“zcytor19” is the previous designation for IL-28 receptor α-subunit, andis shown in SEQ ID NOs:11, 12, 13, 14, 15, 16. The polynucleotides andpolypeptides are described in PCT application WO 02/20569 on behalf ofSchering, Inc., and WO 02/44209 assigned to ZymoGenetics, Inc andincorporated herein by reference. “IL-28 receptor” denotes the IL-28α-subunit and CRF2-4 subunit forming a heterodimeric receptor.

In one aspect, the present invention provides methods for treating viralinfections comprising administering to a mammal with a viral infection atherapeutically effective amount of a polypeptide comprising an aminoacid sequence that has at least 95% identity to amino acid residues ofSEQ ID NO:134, wherein after administration of the polypeptide the viralinfection level is reduced. In other embodiments, the methods compriseadministering a polypeptide comprising an amino acid sequence selectedfrom the group of SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32,34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and 136. Thepolypeptide may optionally comprise at least 15, at least 30, at least45, or at least 60 sequential amino acids of an amino acid sequenceselected from the group of SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28,30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and136. In another aspect, the viral infection can optionally cause liverinflammation, wherein administering a therapeutically effective amountof a polypeptide reduces the liver inflammation. In other embodiments,the polypeptide is conjugated to a polyalkyl oxide moiety, such aspolyethylene glycol (PEG), or F_(c), or human albumin. The PEG may beN-terminally conjugated to the polypeptide and may comprise, forinstance, a 20 kD or 30 kD monomethoxy-PEG propionaldehyde. In anotherembodiment, a reduction in the viral infection level is measured as adecrease in viral load, an increase in antiviral antibodies, a decreasein serological levels of alanine aminotransferase or histologicalimprovement. In another embodiment, the mammal is a human. In anotherembodiment, the present invention provides that the viral infection is ahepatitis B viral infection and/or a hepatitis C viral infection. Inanother embodiment, the polypeptide may be given prior to, concurrentwith, or subsequent to, at least one additional antiviral agent selectedfrom the group of Interferon alpha, Interferon beta, Interferon gamma,Interferon omega, protease inhibitor, RNA or DNA polymerase inhibitor,nucleoside analog, antisense inhibitor, and combinations thereof. Thepolypeptide may be administered intravenously, intraperitoneally,intrathecally, intramuscularly, subcutaneously, orally, intranasally, orby inhalation.

In one aspect, the present invention provides methods for treating viralinfections comprising administering to a mammal with a viral infection atherapeutically effective amount of a composition comprising apolypeptide comprising an amino acid sequence that has at least 95%identity to amino acid residues of SEQ ID NO:134, and a pharmaceuticallyacceptable vehicle, wherein after administration of the composition theviral infection level is reduced. In other embodiments, the methodscomprise administering composition comprising the polypeptide comprisingan amino acid sequence as shown in SEQ ID NOs:2, 4, 6, 18, 20, 22, 24,26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,and/or 136. The polypeptide may optionally comprise at least 15, atleast 30, at least 45, or at least 60 sequential amino acids of an aminoacid sequence as shown in SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28,30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and/or136. In other embodiments, the polypeptide is conjugated to a polyalkyloxide moiety, such as PEG, or F_(c), or human albumin. The PEG may beN-terminally conjugated to the polypeptide and may comprise, forinstance, a 20 kD or 30 kD monomethoxy-PEG propionaldehyde. In anotherembodiment, a reduction in the viral infection level is measured as adecrease in viral load, an increase in antiviral antibodies, a decreasein serological levels of alanine aminotransferase or histologicalimprovement. In another embodiment, the mammal is a human. In anotherembodiment, the present invention provides that the viral infection is ahepatitis B virus infection or a hepatitis C virus infection. In anotherembodiment, the composition may further include or, be given prior toor, be given concurrent with, or be given subsequent to, at least oneadditional antiviral agent selected from the group of Interferon alpha,Interferon beta, Interferon gamma, Interferon omega, protease inhibitor,RNA or DNA polymerase inhibitor, nucleoside analog, antisense inhibitor,and combinations thereof. The composition may be administeredintravenously, intraperitoneally, intrathecally, intramuscularly,subcutaneously, orally, intranasally, or by inhalation.

In one aspect, the present invention provides methods for treating viralinfections comprising administering to a mammal with a viral infectioncausing liver inflammation a therapeutically effective amount of acomposition comprising a polypeptide comprising an amino acid sequencethat has at least 95% identity to amino acid residues of SEQ ID NO:134,and a pharmaceutically acceptable vehicle, wherein after administrationof the composition the viral infection level or liver inflammation isreduced. In other embodiments, the methods comprise administeringcomposition comprising the polypeptide comprising an amino acid sequenceas shown in SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, and/or 136. The polypeptidemay optionally comprise at least 15, at least 30, at least 45, or atleast 60 sequential amino acids of an amino acid sequence as shown inSEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, and/or 136. In other embodiments, thepolypeptide is conjugated to a polyalkyl oxide moiety, such as PEG, orF_(c), or human albumin. The PEG may be N-terminally conjugated to thepolypeptide and may comprise, for instance, a 20 kD or 30 kDmonomethoxy-PEG propionaldehyde. In another embodiment, a reduction inthe viral infection level is measured as a decrease in viral load, anincrease in antiviral antibodies, a decrease in serological levels ofalanine aminotransferase or histological improvement. In anotherembodiment, the mammal is a human. In another embodiment, the presentinvention provides that the viral infection is a hepatitis B virusinfection or a hepatitis C virus infection. In another embodiment, thecomposition may further include or, be given prior to or, be givenconcurrent with, or be given subsequent to, at least one additionalantiviral agent selected from the group of Interferon alpha, Interferonbeta, Interferon gamma, Interferon omega, protease inhibitor, RNA or DNApolymerase inhibitor, nucleoside analog, antisense inhibitor, andcombinations thereof. The composition may be administered intravenously,intraperitoneally, intrathecally, intramuscularly, subcutaneously,orally, intranasally, or by inhalation.

In another aspect, the present invention provides methods for treatingliver inflammation comprising administering to a mammal in need thereofa therapeutically effective amount of a polypeptide comprising an aminoacid sequence that has at least 95% identity to amino acid residues ofSEQ ID NO:134, wherein after administration of the polypeptide the liverinflammation is reduced. In one embodiment, the invention provides thatthe polypeptide comprises an amino acid sequence as shown in SEQ IDNOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, and/or 136. The polypeptide may optionallycomprise at least 15, at least 30, at least 45, or at least 60sequential amino acids of an amino acid sequence as shown in SEQ IDNOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, and/or 136. In another embodiment, thepolypeptide is conjugated to a polyalkyl oxide moiety, such as PEG, orhuman albumin, or F_(c). The PEG may be N-terminally conjugated to thepolypeptide and may comprise, for instance, a 20 kD or 30 kDmonomethoxy-PEG propionaldehyde. In another embodiment, the presentinvention provides that the reduction in the liver inflammation ismeasured as a decrease in serological level of alanine aminotransferaseor histological improvement. In another embodiment, the mammal is ahuman. In another embodiment, the liver inflammation is associated witha hepatitis C viral infection or a hepatitis B viral infection. Inanother embodiment, the polypeptide may be given prior to, concurrentwith, or subsequent to, at least one additional antiviral agent selectedfrom the group of Interferon alpha, Interferon beta, Interferon gamma,Interferon omega, protease inhibitor, RNA or DNA polymerase inhibitor,nucleoside analog, antisense inhibitor, and combinations thereof. Thepolypeptide may be administered intravenously, intraperitoneally,intrathecally, intramuscularly, subcutaneously, orally, intranasally, orby inhalation.

In another aspect, the present invention provides methods for treatingliver inflammation comprising administering to a mammal in need thereofa therapeutically effective amount of a composition comprising apolypeptide comprising an amino acid sequence that has at least 95%identity to amino acid residues of SEQ ID NO:134, wherein afteradministration of the polypeptide the liver inflammation is reduced. Inone embodiment, the invention provides that the polypeptide comprises anamino acid sequence as shown in SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26,28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and/or136. The polypeptide may optionally comprise at least 15, at least 30,at least 45, or at least 60 sequential amino acids of an amino acidsequence as shown in SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32,34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and/or 136. Inanother embodiment, the polypeptide is conjugated to a polyalkyl oxidemoiety, such as PEG, or human albumin, or F_(c). The PEG may beN-terminally conjugated to the polypeptide and may comprise, forinstance, a 20 kD or 30 kD monomethoxy-PEG propionaldehyde. In anotherembodiment, the present invention provides that the reduction in theliver inflammation is measured as a decrease in serological level ofalanine aminotransferase or histological improvement. In anotherembodiment, the mammal is a human. In another embodiment, the liverinflammation is associated with a hepatitis C virus infection or ahepatitis B virus infection. In another embodiment, the composition mayfurther include or, be given prior to or, be given concurrent with, orbe given subsequent to, at least one additional antiviral agent selectedfrom the group of Interferon alpha, Interferon beta, Interferon gamma,Interferon omega, protease inhibitor, RNA or DNA polymerase inhibitor,nucleoside analog, antisense inhibitor, and combinations thereof. Thecomposition may be administered intravenously, intraperitoneally,intrathecally, intramuscularly, subcutaneously, orally, intranasally, orby inhalation.

In another aspect, the present invention provides methods of treating aviral infection comprising administering to an immunocompromised mammalwith an viral infection a therapeutically effective amount of apolypeptide comprising an amino acid sequence that has at least 95%identity to amino acid residues of SEQ ID NO:134, wherein afteradministration of the polypeptide the viral infection is reduced. Inanother embodiment, the polypeptide comprises an amino acid sequence asshown in SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, and/or 136. The polypeptidemay optionally comprise at least 15, at least 30, at least 45, or atleast 60 sequential amino acids of an amino acid sequence as shown inSEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, and/or 136. In another embodiment,the polypeptide is conjugated to a polyalkyl oxide moiety, such as PEG,or human albumin, or F_(c). The PEG may be N-terminally conjugated tothe polypeptide and may comprise, for instance, a 20 kD or 30 kDmonomethoxy-PEG propionaldehyde. In another embodiment, a reduction inthe viral infection level is measured as a decrease in viral load, anincrease in antiviral antibodies, a decrease in serological levels ofalanine aminotransferase or histological improvement. In anotherembodiment, the mammal is a human. In another embodiment, the presentinvention provides that the viral infection is a hepatitis B virusinfection or a hepatitis C virus infection. In another embodiment, thepolypeptide may be given prior to, concurrent with, or subsequent to, atleast one additional antiviral agent selected from the group ofInterferon alpha, Interferon beta, Interferon gamma, Interferon omega,protease inhibitor, RNA or DNA polymerase inhibitor, nucleoside analog,antisense inhibitor, and combinations thereof. The polypeptide may beadministered intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, orally, intranasally, or by inhalation.

In another aspect, the present invention provides methods of treatingliver inflammation comprising administering to an immunocompromisedmammal with liver inflammation a therapeutically effective amount of apolypeptide comprising an amino acid sequence that has at least 95%identity to amino acid residues of SEQ ID NO:134, wherein afteradministration of the polypeptide the liver inflammation is reduced. Inanother embodiment, the polypeptide comprises an amino acid sequence asshown in SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, and/or 136. The polypeptidemay optionally comprise at least 15, at least 30, at least 45, or atleast 60 sequential amino acids of an amino acid sequence as shown inSEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, and/or 136. In another embodiment,the polypeptide is conjugated to a polyalkyl oxide moiety, such as PEG,or human albumin, or F_(c). The PEG may be N-terminally conjugated tothe polypeptide and may comprise, for instance, a 20 kD or 30 kDmonomethoxy-PEG propionaldehyde. In another embodiment, a reduction inthe liver inflammation level is measured as a decrease in serologicallevels of alanine aminotransferase or histological improvement. Inanother embodiment, the mammal is a human. In another embodiment, thepresent invention provides that the viral infection is a hepatitis Bvirus infection or a hepatitis C virus infection. In another embodiment,the mammal is a human. In another embodiment, the present inventionprovides that the viral infection is a hepatitis B virus infection or ahepatitis C virus infection. In another embodiment, the polypeptide maybe given prior to, concurrent with, or subsequent to, at least oneadditional antiviral agent selected from the group of Interferon alpha,Interferon beta, Interferon gamma, Interferon omega, protease inhibitor,RNA or DNA polymerase inhibitor, nucleoside analog, antisense inhibitor,and combinations thereof. The polypeptide may be administeredintravenously, intraperitoneally, intrathecally, intramuscularly,subcutaneously, orally, intranasally, or by inhalation.

The discovery of a new family of interferon-like molecules waspreviously described in PCT applications, PCT/US01/21087 andPCT/US02/12887, and Sheppard et al., Nature Immunol. 4:63-68, 2003; U.S.Patent Application Ser. Nos. 60/493,194 and 60/551,841; all incorporatedby reference herein. This new family includes molecules designatedzcyto20, zcyto21, zcyto22, zcyto24, zcyto25, where zcyto20, 21, and 22are human sequences, and zcyto24 and 25 are mouse sequences. HUGOdesignations have been assigned to the interferon-like proteins. Zcyto20has been designated IL-28A, zycto22 has been designated IL-28B, zycto21has been designated IL-29. Kotenko et al., Nature Immunol. 4:69-77,2003, have identified IL-28A as IFNλ2, IL-28B as IFNλ3, and IL-29 asIFNλ1. The receptor for these proteins, originally designated zcytor19(SEQ ID NOs:11 and 12), has been designated as IL-28RA by HUGO. Whenreferring to “IL-28”, the term shall mean both IL-28A and IL-28B.

The present invention provides methods for using IL-28 and IL-29 as anantiviral agent in a broad spectrum of viral infections. In certainembodiments, the methods include using IL-28 and IL-29 in viralinfections that are specific for liver, such as hepatitis. Furthermore,data indicate that IL-28 and IL-29 exhibit these antiviral activitieswithout some of the toxicities associated with the use of IFN therapyfor viral infection. One of the toxicities related to type I interferontherapy is myelosuppression. This is due to type I interferonssuppression of bone marrow progenitor cells. Because IL-29 does notsignificantly suppress bone marrow cell expansion or B cellproliferation as is seen with IFN-α, IL-29 will have less toxicityassociated with treatment. Similar results would be expected with IL-28Aand IL-28B.

IFN-α may be contraindicated in some patients, particularly when dosessufficient for efficacy have some toxicity or myelosuppressive effects.Examples of patients for which IFN is contraindicated can include (1)patients given previous immunosuppressive medication, (2) patients withHIV or hemophilia, (3) patients who are pregnant, (4) patients with acytopenia, such as leukocyte deficiency, neutropenia, thrombocytopenia,and (5) patients exhibiting increased levels of serum liver enzymes.Moreover, IFN therapy is associated with symptoms that are characterizedby nausea, vomiting, diarrhea and anorexia. The result being that somepopulations of patients will not tolerate IFN therapy, and IL-28A,IL-28B, and IL-29 can provide an alternative therapy for some of thosepatients.

The methods of the present invention comprise administering atherapeutically effective amount of an IL-28A, IL-28B, and/or IL-29polypeptide of the present invention that have retained some biologicalactivity associated with IL-28A, IL-28B or IL-29, alone or incombination with other biologics or pharmaceuticals. The presentinvention provides methods of treating a mammal with a chronic or acuteviral infection, causing liver inflammation, thereby reducing the viralinfection or liver inflammation. In another aspect, the presentinvention provides methods of treating liver specific diseases, inparticular liver disease where viral infection is in part an etiologicagent. These methods are based on the discovery that IL-28 and IL-29have antiviral activity on hepatic cells.

As stated above, the methods of the present invention provideadministering a therapeutically effective amount of an IL-28A, IL-28B,and/or IL-29 polypeptide of the present invention that have retainedsome biological activity associated with IL-28A, IL-28B or IL-29, aloneor in combination with other biologics or pharmaceuticals. The presentinvention provides methods of treatment of a mammal with a viralinfection selected from the group consisting of hepatitis A, hepatitisB, hepatitis C, and hepatitis D. Other aspects of the present inventionprovide methods for using IL-28 or IL-29 as an antiviral agent in viralinfections selected from the group consisting of respiratory syncytialvirus, herpes virus, Epstein-Barr virus, norovirus, influenza virus,adenovirus, parainfluenza virus, rhino virus, coxsackie virus, vacciniavirus, west nile virus, severe acute respiratory syndrome, dengue virus,Venezuelan equine encephalitis virus, pichinde virus and polio virus. Incertain embodiments, the mammal can have either a chronic or acute viralinfection.

In another aspect, the methods of the present invention also include amethod of treating a viral infection comprising administering atherapeutically effective amount of IL-28A, IL-28B, and/or IL-29polypeptide of the present invention that have retained some biologicalactivity associated with IL-28A, IL-28B or IL-29, alone or incombination with other biologics or pharmaceuticals, to animmunompromised mammal with a viral infection, thereby reducing theviral infection, such as is described above. All of the above methods ofthe present invention can also comprise the administration of zcyto24 orzcyto25 as well.

IL-28 and IL-29 are known to have an odd number of cysteines (PCTapplication WO 02/086087 and Sheppard et al., supra.) Expression ofrecombinant IL-28 and IL-29 can result in a heterogeneous mixture ofproteins composed of intramolecular disulfide bonding in multipleconformations. The separation of these forms can be difficult andlaborious. It is therefore desirable to provide IL-28 and IL-29molecules having a single intramolecular disulfide bonding pattern uponexpression and methods for refolding and purifying these preparations tomaintain homogeneity. Thus, the present invention provides forcompositions and methods to produce homogeneous preparations of IL-28and IL-29.

The present invention provides polynucleotide molecules, including DNAand RNA molecules, that encode Cysteine mutants of IL-28 and IL-29 thatresult in expression of a recombinant IL-28 or IL-29 preparation that isa homogeneous preparation. For the purposes of this invention, ahomogeneous preparation of IL-28 and IL-29 is a preparation in whichcomprises at least 98% of a single intramolecular disulfide bondingpattern in the purified polypeptide. In other embodiments, the singledisulfide conformation in a preparation of purified polypeptide is at99% homogeneous. In general, these Cysteine mutants will maintain somebiological activity of the wildtype IL-28 or IL-29, as described herein.For example, the molecules of the present invention can bind to theIL-28 receptor with some specificity. Generally, a ligand binding to itscognate receptor is specific when the K_(D) falls within the range of100 nM to 100 pM. Specific binding in the range of 100 mM to 10 nM K_(D)is low affinity binding. Specific binding in the range of 2.5 pM to 100pM K_(D) is high affinity binding. In another example, biologicalactivity of IL-28 or IL-29 Cysteine mutants is present when themolecules are capable of some level of antiviral activity associatedwith wildtype IL-28 or IL-29. Determination of the level of antiviralactivity is described in detail herein.

An IL-28A gene encodes a polypeptide of 200 amino acids, as shown in SEQID NO:2. The signal sequence for IL-28A comprises amino acid residue 1(Met) through amino acid residue 21 (Ala) of SEQ ID NO:2. The maturepeptide for IL-28A begins at amino acid residue 22 (Val). A variantIL-28A gene encodes a polypeptide of 200 amino acids, as shown in SEQ IDNO:18. The signal sequence for IL-28A can be predicted as comprisingamino acid residue −25 (Met) through amino acid residue −1 (Ala) of SEQID NO:18. The mature peptide for IL-28A begins at amino acid residue 1(Val). IL-28A helices are predicted as follow: helix A is defined byamino acid residues 31 (Ala) to 45 (Leu); helix B by amino acid residues58 (Thr) to 65 (Gln); helix C by amino acid residues 69 (Arg) to 86(Ala); helix D by amino acid residues 95 (Val) to 114 (Ala); helix E byamino acid residues 126 (Thr) to 142 (Lys); and helix F by amino acidresidues 148 (Cys) to 169 (Ala); as shown in SEQ ID NO:18. When apolynucleotide sequence encoding the mature polypeptide is expressed ina prokaryotic system, such as E. coli, a secretory signal sequence maynot be required and an N-terminal Met may be present, resulting inexpression of a polypeptide such as, for instance, as shown in SEQ IDNO:36.

IL-28A polypeptides of the present invention also include a mutation atthe second cysteine, C2, of the mature polypeptide. For example, C2 fromthe N-terminus of the polypeptide of SEQ ID NO:18 is the cysteine atamino acid position 48 (position 49, additional N-terminal Met, ifexpressed in E coli, see, for example, SEQ ID NO:36). This secondcysteine (of which there are seven, like IL-28B) or C2 of IL-28A can bemutated, for example, to a serine, alanine, threonine, valine, orasparagine. IL-28A C2 mutant molecules of the present invention include,for example, polynucleotide molecules as shown in SEQ ID NOs:23 and 25,including DNA and RNA molecules, that encode IL-28A C2 mutantpolypeptides as shown in SEQ ID NOs:24 and 26, respectively.

In addition to the IL-28A C2 mutants, the present invention alsoincludes IL-28A polypeptides comprising a mutation at the third cysteineposition, C3, of the mature polypeptide. For example, C3 from theN-terminus of the polypeptide of SEQ ID NO:18, is the cysteine atposition 50, (position 51, additional N-terminal Met, if expressed in Ecoli, see, for example, SEQ ID NO:36). IL-28A C3 mutant molecules of thepresent invention include, for example, polynucleotide molecules asshown in SEQ ID NOs:27 and 29, including DNA and RNA molecules, thatencode IL-28A C3 mutant polypeptides as shown in SEQ ID NOs:28 and 30,respectively (PCT publication WO 03/066002 (Kotenko et al.)).

The IL-28A polypeptides of the present invention include, for example,SEQ ID NOs:2, 18, 24, 26, 28, 30, 36, and biologically active mutants,fusions, variants and fragments thereof which are encoded by IL-28Apolynucleotide molecules as shown in SEQ ID NOs:1, 17, 23, 25, 27, 29,and 35, respectively.

An IL-29 gene encodes a polypeptide of 200 amino acids, as shown in SEQID NO:4. The signal sequence for IL-29 comprises amino acid residue 1(Met) through amino acid residue 19 (Ala) of SEQ ID NO:4. The maturepeptide for IL-29 begins at amino acid residue 20 (Gly). IL-29 has beendescribed in published PCT application WO 02/02627. A variant IL-29 geneencodes a polypeptide of 200 amino acids, as shown in, for example, SEQID NO:20, where amino acid residue 188 (or amino acid residue 169 of themature polypeptide which begins from amino acid residue 20 (Gly)) is Asninstead of Asp. The present invention also provides a variant IL-29 genewherein the mature polypeptide has a Thr at amino acid residue 10substituted with a Pro, such as, for instance, SEQ ID NOs:54, 56, 58,60, 62, 64, 66, and 68, which are encoded by the polynucleotidesequences as shown in SEQ ID NOs:53, 55, 57, 59, 61, 63, 65, and 67,respectively. The present invention also provides a variant IL-29 genewherein the mature polypeptide has a Gly at amino acid residue 18substituted with an Asp, such as, for instance, SEQ ID NOs:70, 72, 74,76, 78, 80, 82, and 84, which are encoded by the polynucleotidesequences as shown in SEQ ID NOs:69, 71, 73, 75, 77, 79, 81, and 83,respectively. The signal sequence for IL-29 can be predicted ascomprising amino acid residue −19 (Met) through amino acid residue −1(Ala) of SEQ ID NO:20. The mature peptide for IL-29 begins at amino acidresidue 1 (Gly) of SEQ ID NO:20. IL-29 has been described in PCTapplication WO 02/02627. IL-29 helices are predicted as follows: helix Ais defined by amino acid residues 30 (Ser) to 44 (Leu); helix B by aminoacid residues 57 (Asn) to 65 (Val); helix C by amino acid residues 70(Val) to 85 (Ala); helix D by amino acid residues 92 (Glu) to 114 (Gln);helix E by amino acid residues 118 (Thr) to 139 (Lys); and helix F byamino acid residues 144 (Gly) to 170 (Leu); as shown in SEQ ID NO:20.When a polynucleotide sequence encoding the mature polypeptide isexpressed in a prokaryotic system, such as E. coli, a secretory signalsequence may not be required and an N-terminal Met may be present,resulting in expression of an IL-29 polypeptide such as, for instance,as shown in SEQ ID NO:38.

IL-29 polypeptides of the present invention also include a mutation atthe fifth cysteine, C5, of the mature polypeptide. For example, C5 fromthe N-terminus of the polypeptide of SEQ ID NO:20, is the cysteine atposition 171, or position 172 (additional N-terminal Met) if expressedin E. coli. (see, for example, SEQ ID NO:38). This fifth cysteine or C5of IL-29 can be mutated, for example, to a serine, alanine, threonine,valine, or asparagine. These IL-29 C5 mutant polypeptides have adisulfide bond pattern of C1(Cys15 of SEQ ID NO:20)/C3(Cys112 of SEQ IDNO:20) and C2(Cys49 of SEQ ID NO:20)/C4(Cys145 of SEQ ID NO:20). IL-29C5 mutant molecules of the present invention include, for example,polynucleotide molecules as shown in SEQ ID NOs:31, 33, 49, 51, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135,including DNA and RNA molecules, that encode IL-29 C5 mutantpolypeptides as shown in SEQ ID NOs:32, 34, 50, 52, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, and 136, respectively.Additional IL-29 C5 mutant molecules of the present invention includepolynucleotide molecules as shown in SEQ ID NOs:53, 55, 61, and 63,including DNA and RNA molecules, that encode IL-29 C5 mutantpolypeptides as shown in SEQ ID NOs:54, 55, 62, and 64, respectively(PCT publication WO 03/066002 (Kotenko et al.)). Additional, IL-29 C5mutant molecules of the present invention include polynucleotidemolecules as shown in SEQ ID NOs:69, 71, 77, and 79, including DNA andRNA molecules, that encode IL-29 C5 mutant polypeptides as shown in SEQID NOs:70, 72, 78, and 80, respectively (PCT publication WO 02/092762(Baum et al.)).

In addition to the IL-29 C5 mutants, the present invention also includesIL-29 polypeptides comprising a mutation at the first cysteine position,C1, of the mature polypeptide. For example, C1 from the N-terminus ofthe polypeptide of SEQ ID NO:20, is the cysteine at position 15, orposition 16 (additional N-terminal Met) if expressed in E. coli (see,for example, SEQ ID NO:38). This first cysteine or C1 of IL-29 can bemutated, for example, to a serine, alanine, threonine, valine, orasparagines. These IL-29 C1 mutant polypeptides will thus have apredicted disulfide bond pattern of C2(Cys49 of SEQ ID NO:20)/C4(Cys145of SEQ ID NO:20) and C3(Cys112 of SEQ ID NO:20)/C5(Cys171 of SEQ IDNO:20). Additional IL-29 C1 mutant molecules of the present inventioninclude polynucleotide molecules as shown in SEQ ID NOs:41, 43, 45, and47, including DNA and RNA molecules, that encode IL-29 C1 mutantpolypeptides as shown in SEQ ID NOs:42, 44, 46, and 48, respectively.Additional IL-29 C1 mutant molecules of the present invention includepolynucleotide molecules as shown in SEQ ID NOs:57, 59, 65, and 67,including DNA and RNA molecules, that encode IL-29 C1 mutantpolypeptides as shown in SEQ ID NOs:58, 60, 66, and 68, respectively(PCT publication WO 03/066002 (Kotenko et al.)). Additional, IL-29 C1mutant molecules of the present invention include polynucleotidemolecules as shown in SEQ ID NOs:73, 75, 81, and 83, including DNA andRNA molecules, that encode IL-29 C1 mutant polypeptides as shown in SEQID NOs:74, 76, 82, and 84, respectively (PCT publication WO 02/092762(Baum et al.)).

The IL-29 polypeptides of the present invention include, for example,SEQ ID NOs:4, 20, 32, 34, 38, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, and biologically activemutants, fusions, variants and fragments thereof which are encoded byIL-29 polynucleotide molecules as shown in SEQ ID NOs:3, 19, 31, 33, 37,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, and 135, respectively, may further include a signal sequenceas shown in SEQ ID NOs:102, 104, 106, or 108. A polynucleotide moleculeencoding the signal sequence polypeptides of SEQ ID NOs:102, 104, 106,and 108 are are shown as SEQ ID NOs:101, 103, 105, and 107,respectively.

An IL-28B gene encodes a polypeptide of 205 amino acids, as shown in SEQID NO:6. The signal sequence for IL-28B comprises amino acid residue 1(Met) through amino acid residue 21 (Ala) of SEQ ID NO:6. The maturepeptide for IL-28B begins at amino acid residue 22 (Val). A variantIL-28B gene encodes a polypeptide of 200 amino acids, as shown in SEQ IDNO:22. The signal sequence for IL-28B can be predicted as comprisingamino acid residue −25 (Met) through amino acid residue −1 (Ala) of SEQID NO:22. The mature peptide for IL-28B begins at amino acid residue 1(Val) of SEQ ID NO:22. IL-28B helices are predicted as follow: helix Ais defined by amino acid residues 31 (Ala) to 45 (Leu); helix B by aminoacid residues 58 (Thr) to 65 (Gln); helix C by amino acid residues 69(Arg) to 86 (Ala); helix D by amino acid residues 95 (Gly) to 114 (Ala);helix E by amino acid residues 126 (Thr) to 142 (Lys); and helix F byamino acid residues 148 (Cys) to 169 (Ala); as shown in SEQ ID NO:22.When a polynucleotide sequence encoding the mature polypeptide isexpressed in a prokaryotic system, such as E. coli, a secretory signalsequence may not be required and an N-terminal Met may present,resulting in expression of a polypeptide such as is shown in SEQ IDNO:40.

IL-28B polypeptides of the present invention also include a mutation atthe second cysteine, C2, of the mature polypeptide. For example, C2 fromthe N-terminus of the polypeptide of SEQ ID NO:22 is the cysteine atamino acid position 48, or position 49 (additional N-terminal Met) ifexpressed in E coli (see, for example, SEQ ID NO:40). This secondcysteine (of which there are seven, like IL-28A) or C2 of IL-28B can bemutated, for example, to a serine, alanine, threonine, valine, orasparagine. IL-28B C2 mutant molecules of the present invention include,for example, polynucleotide molecules as shown in SEQ ID NOs:85, and 87,including DNA and RNA molecules, that encode IL-28B C2 mutantpolypeptides as shown in SEQ ID NOs:86 and 88, respectively. AdditionalIL-28B C2 mutant molecules of the present invention includepolynucleotide molecules as shown in SEQ ID NOs:93 and 95 including DNAand RNA molecules, that encode IL-28 C2 mutant polypeptides as shown inSEQ ID NOs:94 and 96, respectively (PCT publication WO 03/066002(Kotenko et al.)).

In addition to the IL-28B C2 mutants, the present invention alsoincludes IL-28B polypeptides comprising a mutation at the third cysteineposition, C3, of the mature polypeptide. For example, C3 from theN-terminus of the polypeptide of SEQ ID NO:22, is the cysteine atposition 50, or position 51 (additional N-terminal Met) if expressed inE. coli (see, for example, SEQ ID NO:40). IL-28B C3 mutant molecules ofthe present invention include, for example, polynucleotide molecules asshown in SEQ ID NOs:89 and 91, including DNA and RNA molecules, thatencode IL-28B C3 mutant polypeptides as shown in SEQ ID NOs:90 and 92,respectively. Additional IL-28B C3 mutant molecules of the presentinvention include polynucleotide molecules as shown in SEQ ID NOs:97 and99 including DNA and RNA molecules, that encode IL-28B C3 mutantpolypeptides as shown in SEQ ID NOs:98 and 100, respectively (PCTpublication WO 03/066002 (Kotenko et al.)).

The IL-28B polypeptides of the present invention include, for example,SEQ ID NOs:6, 22, 40, 86, 88, 90, 92, 94, 96, 98, 100, and biologicallyactive mutants, fusions, variants and fragments thereof which areencoded by IL-28B polynucleotide molecules as shown in SEQ ID NOs:5, 21,39, 85, 87, 89, 91, 93, 95, 97, and 99, respectively.

Zcyto24 gene encodes a polypeptide of 202 amino acids, as shown in SEQID NO:8. Zcyto24 secretory signal sequence comprises amino acid residue1 (Met) through amino acid residue 28 (Ala) of SEQ ID NO:8. Analternative site for cleavage of the secretory signal sequence can befound at amino acid residue 24 (Thr). The mature polypeptide comprisesamino acid residue 29 (Asp) to amino acid residue 202 (Val).

Zcyto25 gene encodes a polypeptide of 202 amino acids, as shown in SEQID NO: 10. Zcyto25 secretory signal sequence comprises amino acidresidue 1 (Met) through amino acid residue 28 (Ala) of SEQ ID NO: 10. Analternative site for cleavage of the secretory signal sequence can befound at amino acid residue 24 (Thr). The mature polypeptide comprisesamino acid residue 29 (Asp) to amino acid residue 202 (Val).

The IL-28 and IL-29 cysteine mutant polypeptides of the presentinvention provided for the expression of a single-disulfide form of theIL-28 or IL-29 molecule. When IL-28 and IL-29 are expressed in E. coli,an N-terminal Methionine is present. SEQ ID NOs:26, and 34, forinstance, show the amino acid residue numbering for IL-28A and IL-29mutants, respectively, when the N-terminal Met is present. Table 1 showsthe possible combinations of intramolecular disulfide bonded cysteinepairs for wildtype IL-28A, IL-28B, and IL-29.

TABLE 1 IL-28A C₁₆-C₁₁₅ C₄₈-C₁₄₈ C₅₀-C₁₄₈ C₁₆₇-C₁₇₄ C₁₆-C₄₈ C₁₆-C₅₀C₄₈-C₁₁₅ C₅₀-C₁₁₅ C₁₁₅-C₁₄₈ SEQ ID NO: 18 Met IL- C₁₇-C₁₁₆ C₄₉-C₁₄₉C₅₁-C₁₄₉₈ C₁₆₈-C₁₇₅ C₁₇-C₄₉ C₁₇-C₅₁ C₄₉-C₁₁₆ C₅₁-C₁₁₆ C₁₁₆-C₁₄₉ 28A SEQID NO: 36 IL-29 C₁₅-C₁₁₂ C₄₉-C₁₄₅ C₁₁₂-C₁₇₁  SEQ ID NO: 20 Met IL-29C₁₆-C₁₁₃ C₅₀-C₁₄₆ C₁₁₃-C₁₇₂  SEQ ID NO: 38 IL-28B C₁₆-C₁₁₅ C₄₈-C₁₄₈C₅₀-C₁₄₈ C₁₆₇-C₁₇₄ C₁₆-C₄₈ C₁₆-C₅₀ C₄₈-C₁₁₅ C₅₀-C₁₁₅ C₁₁₅-C₁₄₈ SEQ IDNO: 22 Met IL-28B C₁₇-C₁₁₆ C₄₉-C₁₄₉ C₅₁-C₁₄₉₈ C₁₆₈-C₁₇₅ C₁₇-C₄₉ C₁₇-C₅₁C₄₉-C₁₁₆ C₅₁-C₁₁₆ C₁₁₆-C₁₄₉ SEQ ID NO: 40

Using methods known in the art, IL-28 or IL-29 polypeptides of thepresent invention can be prepared as monomers or multimers; glycosylatedor non-glycosylated; pegylated or non-pegylated; fusion proteins; andmay or may not include an initial methionine amino acid residue. IL-28or IL-29 polypeptides can be conjugated to acceptable water-solublepolymer moieties for use in therapy. Conjugation of interferons, forexample, with water-soluble polymers has been shown to enhance thecirculating half-life of the interferon, and to reduce theimmunogenicity of the polypeptide (see, for example, Nieforth et al.,Clin. Pharmacol. Ther. 59:636 (1996), and Monkarsh et al., Anal.Biochem. 247:434 (1997)).

Suitable water-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinylpyrrolidone)PEG, tresyl monomethoxy PEG, monomethoxy-PEGpropionaldehyde, PEG propionaldehyde, bis-succinimidyl carbonate PEG,propylene glycol homopolymers, a polypropylene oxide/ethylene oxideco-polymer, polyoxyethylated polyols (e.g., glycerol), monomethoxy-PEGbutyraldehyde, PEG butyraldehyde, monomethoxy-PEG acetaldehyde, PEGacetaldehyde, methoxyl PEG-succinimidyl propionate, methoxylPEG-succinimidyl butanoate, polyvinyl alcohol, dextran, cellulose, orother carbohydrate-based polymers. Suitable PEG may have a molecularweight from about 600 to about 60,000, including, for example, 5,000,12,000, 20,000, 30,000, 40,000, and 50,000, which can be linear orbranched. A IL-28 or IL-29 conjugate can also comprise a mixture of suchwater-soluble polymers.

One example of an IL-28 or IL-29 conjugate comprises an IL-28 or IL-29moiety and a polyalkyl oxide moiety attached to the N-terminus of theIL-28 or IL-29 moiety. PEG is one suitable polyalkyl oxide. As anillustration, IL-28 or IL-29 can be modified with PEG, a process knownas “PEGylation.” PEGylation of an IL-28 or IL-29 can be carried out byany of the PEGylation reactions known in the art (see, for example, EP 0154 316, Delgado et al., Critical Reviews in Therapeutic Drug CarrierSystems 9:249 (1992), Duncan and Spreafico, Clin. Pharmacokinet. 27:290(1994), and Francis et al., Int J Hematol 68:1 (1998)). For example,PEGylation can be performed by an acylation reaction or by an alkylationreaction with a reactive polyethylene glycol molecule. In an alternativeapproach, IL-28 or IL-29 conjugates are formed by condensing activatedPEG, in which a terminal hydroxy or amino group of PEG has been replacedby an activated linker (see, for example, Karasiewicz et al., U.S. Pat.No. 5,382,657).

PEGylation by acylation typically requires reacting an active esterderivative of PEG with an IL-28 or IL-29 polypeptide. An example of anactivated PEG ester is PEG esterified to N-hydroxysuccinimide. As usedherein, the term “acylation” includes the following types of linkagesbetween IL-28 or IL-29 and a water-soluble polymer: amide, carbamate,urethane, and the like. Methods for preparing PEGylated IL-28 or IL-29by acylation will typically comprise the steps of (a) reacting an IL-28or IL-29 polypeptide with PEG (such as a reactive ester of an aldehydederivative of PEG) under conditions whereby one or more PEG groupsattach to IL-28 or IL-29, and (b) obtaining the reaction product(s).Generally, the optimal reaction conditions for acylation reactions willbe determined based upon known parameters and desired results. Forexample, the larger the ratio of PEG: IL-28 or IL-29, the greater thepercentage of polyPEGylated IL-28 or IL-29 product.

PEGylation by alkylation generally involves reacting a terminalaldehyde, e.g., propionaldehyde, butyraldehyde, acetaldehyde, and thelike, derivative of PEG with IL-28 or IL-29 in the presence of areducing agent. PEG groups are preferably attached to the polypeptidevia a —CH₂—NH₂ group.

Derivatization via reductive alkylation to produce a monoPEGylatedproduct takes advantage of the differential reactivity of differenttypes of primary amino groups available for derivatization. Typically,the reaction is performed at a pH that allows one to take advantage ofthe pKa differences between the C-amino groups of the lysine residuesand the α-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water-soluble polymer thatcontains a reactive group such as an aldehyde, to a protein iscontrolled. The conjugation with the polymer occurs predominantly at theN-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups.

Reductive alkylation to produce a substantially homogenous population ofmonopolymer IL-28 or IL-29 conjugate molecule can comprise the steps of:(a) reacting an IL-28 or IL-29 polypeptide with a reactive PEG underreductive alkylation conditions at a pH suitable to permit selectivemodification of the α-amino group at the amino terminus of the IL-28 orIL-29, and (b) obtaining the reaction product(s). The reducing agentused for reductive alkylation should be stable in aqueous solution andpreferably be able to reduce only the Schiff base formed in the initialprocess of reductive alkylation. Preferred reducing agents includesodium borohydride, sodium cyanoborohydride, dimethylamine borane,trimethylamine borane, and pyridine borane.

For a substantially homogenous population of monopolymer IL-28 or IL-29conjugates, the reductive alkylation reaction conditions are those thatpermit the selective attachment of the water-soluble polymer moiety tothe N-terminus of IL-28 or IL-29. Such reaction conditions generallyprovide for pKa differences between the lysine amino groups and theα-amino group at the N-terminus. The pH also affects the ratio ofpolymer to protein to be used. In general, if the pH is lower, a largerexcess of polymer to protein will be desired because the less reactivethe N-terminal α-group, the more polymer is needed to achieve optimalconditions. If the pH is higher, the polymer: IL-28 or IL-29 need not beas large because more reactive groups are available. Typically, the pHwill fall within the range of 3-9, or 3-6. Another factor to consider isthe molecular weight of the water-soluble polymer. Generally, the higherthe molecular weight of the polymer, the fewer number of polymermolecules which may be attached to the protein. For PEGylationreactions, the typical molecular weight is about 2 kDa to about 100 kDa,about 5 kDa to about 50 kDa, or about 12 kDa to about 40 kDa. The molarratio of water-soluble polymer to IL-28 or IL-29 will generally be inthe range of 1:1 to 100:1. Typically, the molar ratio of water-solublepolymer to IL-28 or IL-29 will be 1:1 to 20:1 for polyPEGylation, and1:1 to 5:1 for monoPEGylation.

General methods for producing conjugates comprising interferon andwater-soluble polymer moieties are known in the art. See, for example,Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al., U.S. Pat.No. 5,738,846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996),Monkarsh et al., Anal. Biochem. 247:434 (1997). PEGylated species can beseparated from unconjugated IL-28 or IL-29 polypeptides using standardpurification methods, such as dialysis, ultrafiltration, ion exchangechromatography, affinity chromatography, size exclusion chromatography,and the like.

The IL-28 or IL-29 polypeptides of the present invention are capable ofspecifically binding the IL-28 receptor and/or acting as an antiviralagent. The binding of IL-28 or 11-29 polypeptides to the IL-28 receptorcan be assayed using established approaches. IL-28 or IL-29 polypeptidescan be iodinated using an iodobead (Pierce, Rockford, Ill.) according tomanufacturer's directions, and the ¹²⁵I-IL-28 or ¹²⁵I-IL-29 can then beused as described below.

In a first approach fifty nanograms of ¹²⁵I-IL-28 or 125I-IL-29 can becombind with 1000 ng of IL-28 receptor human IgG fusion protein, in thepresence or absence of possible binding competitors including unlabeledcysteine mutant IL-28, cysteine mutant IL-29, IL-28, or IL-29. The samebinding reactions would also be performed substituting other cytokinereceptor human IgG fusions as controls for specificity. Followingincubation at 4° C., protein-G (Zymed, SanFransisco, Calif.) is added tothe reaction, to capture the receptor-IgG fusions and any proteins boundto them, and the reactions are incubated another hour at 4° C. Theprotein-G sepharose is then collected, washed three times with PBS and¹²⁵I-IL-28 or ¹²⁵I-IL-29 bound is measure by gamma counter (PackardInstruments, Downers Grove, Ill.).

In a second approach, the ability of molecules to inhibit the binding of¹²⁵I-IL-28 or ¹²⁵I-IL-29 to plate bound receptors can be assayed. Afragment of the IL-28 receptor, representing the extracellular, ligandbinding domain, can be adsorbed to the wells of a 96 well plate byincubating 100 μl of 1 g/mL solution of receptor in the plate overnight.In a second form, a receptor-human IgG fusion can be bound to the wellsof a 96 well plate that has been coated with an antibody directedagainst the human IgG portion of the fusion protein. Following coatingof the plate with receptor the plate is washed, blocked with SUPERBLOCK(Pierce, Rockford, Ill.) and washed again. Solutions containing a fixedconcentration of 125I-IL-28 or 125I-IL-29 with or without increasingconcentrations of potential binding competitors including, Cysteinemutant IL-28, cysteine mutant IL-29, IL-28 and IL-29, and 100 μl of thesolution added to appropriate wells of the plate. Following a one hourincubation at 4° C. the plate is washed and the amount ¹²⁵I-IL-28 or¹²⁵I-IL-29 bound determined by counting (Topcount, Packard Instruments,Downers grove, Ill.). The specificity of binding of ¹²⁵I-IL-28 or¹²⁵I-IL-29 can be defined by receptor molecules used in these bindingassays as well as by the molecules used as inhibitors.

In addition to pegylation, human albumin can be coupled to an IL-28 orIL-29 polypeptide of the present invention to prolong its half-life.Human albumin is the most prevalent naturally occurring blood protein inthe human circulatory system, persisting in circulation in the body forover twenty days. Research has shown that therapeutic proteinsgenetically fused to human albumin have longer half-lives. An IL28 orIL29 albumin fusion protein, like pegylation, may provide patients withlong-acting treatment options that offer a more convenientadministration schedule, with similar or improved efficacy and safetycompared to currently available treatments (U.S. Pat. No. 6,165,470;Syed et al., Blood, 89(9):3243-3253 (1997); Yeh et al., Proc. Natl.Acad. Sci. USA, 89:1904-1908 (1992); and Zeisel et al., Horm. Res.,37:5-13 (1992)).

Like the aforementioned peglyation and human albumin, an Fc portion ofthe human IgG molecule can be fused to a polypeptide of the presentinvention. The resultant fusion protein may have an increasedcirculating half-life due to the Fc moiety (U.S. Pat. No. 5,750,375,U.S. Pat. No. 5,843,725, U.S. Pat. No. 6,291,646; Barouch et al.,Journal of Immunology, 61:1875-1882 (1998); Barouch et al., Proc. Natl.Acad. Sci. USA, 97(8):4192-4197 (Apr. 11, 2000); and Kim et al.,Transplant Proc., 30(8):4031-4036 (December 1998)).

IL-28A, IL-29, IL-28B, zcyto24 and zcyto25, each have been shown to forma complex with the orphan receptor designated zcytor19 (IL-28RA).IL-28RA is described in a commonly assigned patent applicationPCT/US01/44808. IL-28B, IL-29, zcyto24, and zcyto25 have been shown tobind or signal through IL-28RA as well, further supporting that IL-28A,IL-29, IL-28B, zcyto24 and zcyto25 are members of the same family ofcytokines. IL-28RA receptor is a class II cytokine receptor. Class IIcytokine receptors usually bind to four-helix-bundle cytokines. Forexample, interleukin-10 and the interferons bind receptors in this class(e.g., interferon-gamma receptor, alpha and beta chains and theinterferon-alpha/beta receptor alpha and beta chains).

Class II cytokine receptors are characterized by the presence of one ormore cytokine receptor modules (CRM) in their extracellular domains.Other class II cytokine receptors include zcytor11 (commonly owned U.S.Pat. No. 5,965,704), CRF2-4 (Genbank Accession No. Z17227), IL-10R(Genbank Accession No.s U00672 and NM_(—)001558), DIRS1, zcytor7(commonly owned U.S. Pat. No. 5,945,511), and tissue factor. IL-28RA,like all known class II receptors except interferon-alpha/beta receptoralpha chain, has only a single class II CRM in its extracellular domain.

Analysis of a human cDNA clone encoding IL-28RA (SEQ ID NO:11) revealedan open reading frame encoding 520 amino acids (SEQ ID NO:12) comprisinga secretory signal sequence (residues 1 (Met) to 20 (Gly) of SEQ IDNO:12) and a mature IL-28RA cytokine receptor polypeptide (residues 21(Arg) to 520 (Arg) of SEQ ID NO:12) an extracellular ligand-bindingdomain of approximately 206 amino acid residues (residues 21 (Arg) to226 (Asn) of SEQ ID NO:12), a transmembrane domain of approximately 23amino acid residues (residues 227 (Trp) to 249 (Trp) of SEQ ID NO:12),and an intracellular domain of approximately 271 amino acid residues(residues 250 (Lys) to 520 (Arg) of SEQ ID NO:12). Within theextracellular ligand-binding domain, there are two fibronectin type IIIdomains and a linker region. The first fibronectin type III domaincomprises residues 21 (Arg) to 119 (Tyr) of SEQ ID NO:12, the linkercomprises residues 120 (Leu) to 124 (Glu) of SEQ ID NO:12, and thesecond fibronectin type III domain comprises residues 125 (Pro) to 223(Pro) of SEQ ID NO:12.

In addition, a human cDNA clone encoding a IL-28RA variant with a 29amino acid deletion was identified. This IL-28RA variant (as shown inSEQ ID NO:13) comprises an open reading frame encoding 491 amino acids(SEQ ID NO:14) comprising a secretory signal sequence (residues 1 (Met)to 20 (Gly) of SEQ ID NO:14) and a mature IL-28RA cytokine receptorpolyptide (residues 21 (Arg) to 491 (Arg) of SEQ ID NO:14) anextracellular ligand-binding domain of approximately 206 amino acidresidues (residues 21 (Arg) to 226 (Asn) of SEQ ID NO:14, atransmembrane domain of approximately 23 amino acid residues (residues227 (Trp) to 249 (Trp) of SEQ ID NO:14), and an intracellular domain ofapproximately 242 amino acid residues (residues 250 (Lys) to 491 (Arg)of SEQ ID NO:14).

A truncated soluble form of the IL-28RA receptor mRNA appears to benaturally expressed. Analysis of a human cDNA clone encoding thetruncated soluble IL-28RA (SEQ ID NO:15) revealed an open reading frameencoding 211 amino acids (SEQ ID NO:16) comprising a secretory signalsequence (residues 1 (Met) to 20 (Gly) of SEQ ID NO:16) and a maturetruncated soluble IL-28RA receptor polyptide (residues 21 (Arg) to 211(Ser) of SEQ ID NO:16) a truncated extracellular ligand-binding domainof approximately 143 amino acid residues (residues 21 (Arg) to 163 (Trp)of SEQ ID NO:16), no transmembrane domain, but an additional domain ofapproximately 48 amino acid residues (residues 164 (Lys) to 211 (Ser) ofSEQ ID NO:16).

IL-28RA is a member of the same receptor subfamily as the class IIcytokine receptors, and receptors in this subfamily may associate toform homodimers that transduce a signal. Several members of thesubfamily (e.g., receptors that bind interferon, IL-10, IL-19, andIL-TIF) combine with a second subunit (termed a β-subunit) to bindligand and transduce a signal. However, in many cases, specificβ-subunits associate with a plurality of specific cytokine receptorsubunits. For example, class II cytokine receptors, such as, zcytor11(U.S. Pat. No. 5,965,704) and CRF2-4 receptor heterodimerize to bind thecytokine IL-TIF (See, WIPO publication WO 00/24758; Dumontier et al., J.Immunol. 164:1814-1819, 2000; Spencer, S D et al., J. Exp. Med.187:571-578, 1998; Gibbs, V C and Pennica Gene 186:97-101, 1997 (CRF2-4cDNA); Xie, M H et al., J. Biol. Chem. 275: 31335-31339, 2000). IL-10βreceptor is believed to be synonymous with CRF2-4 (Dumoutier, L. et al.,Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000; Liu Y et al., J Immunol.152; 1821-1829, 1994 (IL-10R cDNA). Therefore, one could expect thatIL-28, IL-29, zcyto24 and zcyto25 would bind either monomeric,homodimeric, heterodimeric and multimeric zcytor19 receptors.Experimental evidence has identified CRF2-4 as the putative bindingpartner for IL-28RA.

Examples of biological activity for molecules used to identify IL-28 orIL-29 molecules that are useful in the methods of the present inventioninclude molecules that can bind to the IL-28 receptor with somespecificity. Generally, a ligand binding to its cognate receptor isspecific when the K_(D) falls within the range of 100 nM to 100 pM.Specific binding in the range of 100 mM to 10 nM K_(D) is low affinitybinding. Specific binding in the range of 2.5 pM to 100 pM K_(D) is highaffinity binding. In another example, biologically active IL-28 or IL-29molecules are capable of some level of antiviral activity associatedwith wildtype IL-28 or IL-29.

The various codons that encode for a given amino acid are set forthbelow in Table 2.

TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by referencing the sequencesdisclosed herein. Variant sequences can be readily tested forfunctionality as described herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit “preferential codon usage.” In general, see,Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of IL-28 or IL-29 RNA. Such tissues andcells are identified by Northern blotting (Thomas, Proc. Natl. Acad.Sci. USA 77:5201, 1980), or by screening conditioned medium from variouscell types for activity on target cells or tissue. Once the activity orRNA producing cell or tissue is identified, total RNA can be preparedusing guanidinium isothiocyanate extraction followed by isolation bycentrifugation in a CsCl gradient (Chirgwin et al., Biochemistry18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNA using themethod of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA using knownmethods. In the alternative, genomic DNA can be isolated.Polynucleotides encoding IL-28 or IL-29 polypeptides are then identifiedand isolated by, for example, hybridization or PCR.

A full-length clones encoding IL-28 or IL-29 can be obtained byconventional cloning procedures. Complementary DNA (cDNA) clones arepreferred, although for some applications (e.g., expression intransgenic animals) it may be preferable to use a genomic clone, or tomodify a cDNA clone to include at least one genomic intron. Methods forpreparing cDNA and genomic clones are well known and within the level ofordinary skill in the art, and include the use of the sequence disclosedherein, or parts thereof, for probing or priming a library. Expressionlibraries can be probed with antibodies to IL-28 receptor fragments, orother specific binding partners.

Those skilled in the art will recognize that the sequence disclosed in,for example, SEQ ID NOs:17, 19 and 21, represent a single allele ofhuman IL-28A, IL-29, and IL28B, respectively, and that allelic variationand alternative splicing are expected to occur. For example, an IL-29variant has been identified where amino acid residue 169 as shown in SEQID NO:19 is an Asn residue whereas its corresponding amino acid residuein SEQ ID NO:4 is an Arg residue, as described in WO 02/086087. Suchallelic variants are included in the present invention. Allelic variantsof IL-28 and IL-29 molecules of the present invention can be cloned byprobing cDNA or genomic libraries from different individuals accordingto standard procedures. Allelic variants of the DNA sequence shown inSEQ ID NOs:17, 19, and 21, including those containing silent mutationsand those in which mutations result in amino acid sequence changes, inaddition to the cysteine mutations, are within the scope of the presentinvention, as are proteins which are allelic variants of SEQ ID NO:18,20, and 22. cDNAs generated from alternatively spliced mRNAs, whichretain the properties of IL-28 or IL-29 polypeptides, are includedwithin the scope of the present invention, as are polypeptides encodedby such cDNAs and mRNAs. Allelic variants and splice variants of thesesequences can be cloned by probing cDNA or genomic libraries fromdifferent individuals or tissues according to standard procedures knownin the art, and mutations to the polynucleotides encoding cysteines orcysteine residues can be introduced as described herein.

Within embodiments of the invention, isolated IL-28 and IL-29-encodingnucleic acid molecules can hybridize under stringent conditions tonucleic acid molecules having the nucleotide sequence selected from thegroup of SEQ ID NOs:1, 3, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, and 135, or to its complementthereof. In general, stringent conditions are selected to be about 5° C.lower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases.

It is well within the abilities of one skilled in the art to adapt theseconditions for use with a particular polynucleotide hybrid. The T_(m)for a specific target sequence is the temperature (under definedconditions) at which 50% of the target sequence will hybridize to aperfectly matched probe sequence. Those conditions which influence theT_(m) include, the size and base pair content of the polynucleotideprobe, the ionic strength of the hybridization solution, and thepresence of destabilizing agents in the hybridization solution. Numerousequations for calculating T_(m) are known in the art, and are specificfor DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences ofvarying length (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Press 1989);Ausubel et al., (eds.), Current Protocols in Molecular Biology (JohnWiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to MolecularCloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such asOLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (PremierBiosoft International; Palo Alto, Calif.), as well as sites on theInternet, are available tools for analyzing a given sequence andcalculating T_(m) based on user defined criteria. Such programs can alsoanalyze a given sequence under defined conditions and identify suitableprobe sequences. Typically, hybridization of longer polynucleotidesequences, >50 base pairs, is performed at temperatures of about 20-25°C. below the calculated T_(m). For smaller probes, <50 base pairs,hybridization is typically carried out at the T_(m) or 5-10° C. belowthe calculated T_(m). This allows for the maximum rate of hybridizationfor DNA-DNA and DNA-RNA hybrids.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. That is, nucleic acid moleculesencoding an IL-28 or IL-29 polypeptide hybridize with a nucleic acidmolecule having the nucleotide sequence selected from the group of SEQID NOs:1, 3, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 99, 109, 111, 113, 115, 117, 119, 121,123, 125, 127, 129, 131, 133, and 135, or its complement thereof, understringent washing conditions, in which the wash stringency is equivalentto 0.5×-2×SSC with 0.1% SDS at 55-65° C., including 0.5×SSC with 0.1%SDS at 55° C., or 2×SSC with 0.1% SDS at 65° C. One of skill in the artcan readily devise equivalent conditions, for example, by substitutingSSPE for SSC in the wash solution.

Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. In other words, nucleic acid molecules encoding a variant ofan IL-28 or IL-29 polypeptide hybridize with a nucleic acid moleculehaving the nucleotide sequence selected from the group of SEQ ID NOs:1,3, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, and 135, or its complement thereof, under highlystringent washing conditions, in which the wash stringency is equivalentto 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., including 0.1×SSC with 0.1%SDS at 50° C., or 0.2×SSC with 0.1% SDS at 65° C.

The present invention also provides isolated IL-28 or IL-29 polypeptidesthat have a substantially similar sequence identity to the polypeptidesof the present invention, for example, selected from the group of SEQ IDNOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, and 136. The term “substantially similarsequence identity” is used herein to denote polypeptides comprising atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%,or greater than 99.5% sequence identity to the amino acid sequencesselected from the group of SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28,30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and136. The present invention also includes polypeptides that comprise anamino acid sequence having at least 80%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%,at least 99%, at least 99.5%, or greater than 99.5% sequence identity toa polypeptide or fragment thereof of the present invention.

The present invention further includes nucleic acid molecules thatencode such polypeptides. The IL-28 and IL-29 polypeptides of thepresent invention are preferably recombinant polypeptides. In anotheraspect, the IL-28 and IL-29 polypeptides of the present invention haveat least 15, at least 30, at least 45, or at least 60 sequential aminoacids. For example, an IL-29 polypeptide of the present inventionrelates to a polypeptide having at least 15, at least 30, at least 45,or at least 60 sequential amino acids to an amino acid sequence selectedfrom the group of SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32,34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and 136. Methodsfor determining percent identity are herein.

The present invention also contemplates variant nucleic acid moleculesthat can be identified using two criteria: a determination of thesimilarity between the encoded polypeptide with the amino acid sequenceselected from the group of SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28,30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and136, and/or a hybridization assay, as described above. Such variantsinclude nucleic acid molecules: (1) that hybridize with a nucleic acidmolecule having the nucleotide sequence selected from the group of SEQID NOs:1, 3, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 99, 109, 111, 113, 115, 117, 119, 121,123, 125, 127, 129, 131, 133, and 135, or complement thereof, understringent washing conditions, in which the wash stringency is equivalentto 0.5×-2×SSC with 0.1% SDS at 55-65° C.; or (2) that encode apolypeptide having at least 80%, at least 90%, at least 95%, at least96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, atleast 99%, at least 99.5%, or greater than 99.5% sequence identity tothe amino acid sequence selected from the group of SEQ ID NOs:2, 4, 6,18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, and 136. Alternatively, variants can be characterized asnucleic acid molecules: (1) that hybridize with a nucleic acid moleculehaving the nucleotide sequence selected from the group of SEQ ID NOs:1,3, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, and 135, or its complement thereof, under highlystringent washing conditions, in which the wash stringency is equivalentto 0.1×-0.2×SSC with 0.1% SDS at 50-65° C.; and (2) that encode apolypeptide having at least 80%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.5%, orgreater than 99.5% sequence identity to the amino acid sequence selectedfrom the group of SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32,34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and 136.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 2 (amino acids are indicated by the standard one-lettercodes).

$\frac{\left( {{Total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {identical}\mspace{14mu} {matches}} \right)}{\begin{bmatrix}{{length}{\mspace{11mu} \;}{of}\mspace{14mu} {the}\mspace{14mu} {longer}\mspace{14mu} {sequence}\mspace{14mu} {plus}\mspace{14mu} {the}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {gaps}} \\{\mspace{14mu} {{introduced}\mspace{14mu} {into}\mspace{14mu} {the}\mspace{14mu} {longer}\mspace{14mu} {sequence}\mspace{14mu} {in}\mspace{14mu} {order}\mspace{14mu} {to}}{\mspace{31mu} \;}} \\{{align}{\mspace{11mu} \;}{the}\mspace{14mu} {two}\mspace{14mu} {sequences}}\end{bmatrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant IL-28 or IL-29. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:2) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62. These parameters can be introducedinto a FASTA program by modifying the scoring matrix file (“SMATRIX”),as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

IL-28 or IL-29 polypeptides with substantially similar sequence identityare characterized as having one or more amino acid substitutions,deletions or additions. These changes are preferably of a minor nature,that is conservative amino acid substitutions (see Table 4) and othersubstitutions that do not significantly affect the folding or activityof the polypeptide; small deletions, typically of one to about 30 aminoacids; and amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue, a small linker peptide of up to about20-25 residues, or an affinity tag. The present invention thus includespolypeptides that comprise a sequence that is at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 97.5%, at least98%, at least 98.5%, at least 99%, at least 99.5%, or greater than 99.5%identical to the corresponding region of SEQ ID NOs:2, 4, 6, 18, 20, 22,24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, and 136. Polypeptides comprising affinity tags can further comprisea proteolytic cleavage site between the IL-28 and IL-29 polypeptide andthe affinity tag. Preferred such sites include thrombin cleavage sitesand factor Xa cleavage sites.

TABLE 4 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

Determination of amino acid residues that comprise regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to alignment of multiple sequences withhigh amino acid or nucleotide identity, secondary structurepropensities, binary patterns, complementary packing and buried polarinteractions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 andCordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general,when designing modifications to molecules or identifying specificfragments determination of structure will be accompanied by evaluatingactivity of modified molecules.

Amino acid sequence changes are made in IL-28 or IL-29 polypeptides soas to minimize disruption of higher order structure essential tobiological activity. For example, where the IL-28 or IL-29 polypeptidecomprises one or more helices, changes in amino acid residues will bemade so as not to disrupt the helix geometry and other components of themolecule where changes in conformation abate some critical function, forexample, binding of the molecule to its binding partners. The effects ofamino acid sequence changes can be predicted by, for example, computermodeling as disclosed above or determined by analysis of crystalstructure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268,1995). Other techniques that are well known in the art compare foldingof a variant protein to a standard molecule (e.g., the native protein).For example, comparison of the cysteine pattern in a variant andstandard molecules can be made. Mass spectrometry and chemicalmodification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same cysteine pattern as thestandard molecule folding would be affected. Another well known andaccepted method for measuring folding is circular dichrosism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructurally similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

A Hopp/Woods hydrophilicity profile of an IL-28 or IL-29 polypeptidesequence selected from the group of SEQ ID NOs:2, 4, 6, 18, 20, 22, 24,26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,and 136, can be generated (Hopp et al., Proc. Natl. Acad. Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquier etal., Protein Engineering 11:153-169, 1998). The profile is based on asliding six-residue window. Buried G, S, and T residues and exposed H,Y, and W residues were ignored. Those skilled in the art will recognizethat hydrophilicity or hydrophobicity will be taken into account whendesigning modifications in the amino acid sequence of an IL-28 or IL-29polypeptide, so as not to disrupt the overall structural and biologicalprofile. Of particular interest for replacement are hydrophobic residuesselected from the group consisting of Val, Leu and Ile or the groupconsisting of Met, Gly, Ser, Ala, Tyr and Trp.

The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between IFN-α and members of the familyof IL-28A, IL-28B, and IL-29 (as shown in Tables 1 and 2). Using methodssuch as “FASTA” analysis described previously, regions of highsimilarity are identified within a family of proteins and used toanalyze amino acid sequence for conserved regions. An alternativeapproach to identifying a variant polynucleotide on the basis ofstructure is to determine whether a nucleic acid molecule encoding apotential variant IL-28 or IL-29 gene can hybridize to a nucleic acidmolecule as discussed above.

Other methods of identifying essential amino acids in the polypeptidesof the present invention are procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Natl. Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant IL-28 and IL-29 molecules are tested forbiological or biochemical activity as disclosed below to identify aminoacid residues that are critical to the activity of the molecule. Seealso, Hilton et al., J. Biol. Chem. 271:4699 (1996).

The present invention also includes functional fragments of IL-28 orIL-29 polypeptides and nucleic acid molecules encoding such functionalfragments. A “functional” IL-28 or IL-29 or fragment thereof as definedherein is characterized by its proliferative or differentiatingactivity, by its ability to induce or inhibit specialized cellfunctions, or by its ability to bind specifically to an anti-IL-28 orIL-29 antibody or IL-28 receptor (either soluble or immobilized). Thespecialized activities of IL-28 or IL-29 polypeptides and how to testfor them are disclosed herein. As previously described herein, IL-28 andIL-29 polypeptides are characterized by a six-helical-bundle. Thus, thepresent invention further provides fusion proteins encompassing: (a)polypeptide molecules comprising one or more of the helices describedabove; and (b) functional fragments comprising one or more of thesehelices. The other polypeptide portion of the fusion protein may becontributed by another helical-bundle cytokine or interferon, such asIFN-α, or by a non-native and/or an unrelated secretory signal peptidethat facilitates secretion of the fusion protein.

The IL-28 or IL-29 polypeptides of the present invention, includingfull-length polypeptides, biologically active fragments, and fusionpolypeptides can be produced according to conventional techniques usingcells into which have been introduced an expression vector encoding thepolypeptide. As used herein, “cells into which have been introduced anexpression vector” include both cells that have been directlymanipulated by the introduction of exogenous DNA molecules and progenythereof that contain the introduced DNA. Suitable host cells are thosecell types that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Techniques for manipulating cloned DNAmolecules and introducing exogenous DNA into a variety of host cells aredisclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, and Ausubel et al., eds., Current Protocols in Molecular Biology,John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding an IL-28 or IL-29 polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers.

To direct an IL-28 or IL-29 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of IL-28 or IL-29, e.g., SEQID NO:119 or SEQ ID NO:121, or may be derived from another secretedprotein (e.g., t-PA; see, U.S. Pat. No. 5,641,655) or synthesized denovo. The secretory signal sequence is operably linked to an IL-28 orIL-29 DNA sequence, i.e., the two sequences are joined in the correctreading frame and positioned to direct the newly synthesized polypeptideinto the secretory pathway of the host cell. Secretory signal sequencesare commonly positioned 5′ to the DNA sequence encoding the polypeptideof interest, although certain signal sequences may be positionedelsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

A wide variety of suitable recombinant host cells includes, but is notlimited to, gram-negative prokaryotic host organisms. Suitable strainsof E. coli include W3110 and mutants-strains thereof (e.g, an OmpTprotease deficient W3110 strain, and an OmpT protease and fhuA deficientW3110 strain), K12-derived strains MM294, TG-1, JM-107, BL21, andUT5600. Other suitable strains include: BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF′, DH5IMCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, ER1647, E. coli K12, E. coli K12 RV308, E. coli K12 C600,E. coliHB101, E. coli K12 C600 R.sub.k-M.sub.k-, E. coli K12 RR1 (see,for example, Brown (ed.), Molecular Biology Labfax (Academic Press1991)). Other gram-negative prokaryotic hosts can include Serratia,Pseudomonas, Caulobacter. Prokaryotic hosts can include gram-positiveorganisms such as Bacillus, for example, B. subtilis and B.thuringienesis, and B. thuringienesis var. israelensis, as well asStreptomyces, for example, S. lividans, S. ambofaciens, S. fradiae, andS. griseofuscus. Suitable strains of Bacillus subtilus include BR151,YB886, MI119, MI120, and B170 (see, for example, Hardy, “BacillusCloning Methods,” in DNA Cloning: A Practical Approach, Glover (ed.)(IRL Press 1985)). Standard techniques for propagating vectors inprokaryotic hosts are well-known to those of skill in the art (see, forexample, Ausubel et al. (eds.), Short Protocols in Molecular Biology,3^(rd) Edition (John Wiley & Sons 1995); Wu et al., Methods in GeneBiotechnology (CRC Press, Inc. 1997)). In one embodiment, the methods ofthe present invention use Cysteine mutant IL-28 or IL-29 expressed inthe W3110 strain, which has been deposited at the American Type CultureCollection (ATCC) as ATCC #27325.

When large scale production of an IL-28 or IL-29 polypeptide using theexpression system of the present invention is required, batchfermentation can be used. Generally, batch fermentation comprises that afirst stage seed flask is prepared by growing E. coli strains expressingan IL-28 or IL-29 polypeptide in a suitable medium in shake flaskculture to allow for growth to an optical density (OD) of between 5 and20 at 600 nm. A suitable medium would contain nitrogen from a source(s)such as ammonium sulfate, ammonium phosphate, ammonium chloride, yeastextract, hydrolyzed animal proteins, hydrolyzed plant proteins orhydrolyzed caseins. Phosphate will be supplied from potassium phosphate,ammonium phosphate, phosphoric acid or sodium phosphate. Othercomponents would be magnesium chloride or magnesium sulfate, ferroussulfate or ferrous chloride, and other trace elements. Growth medium canbe supplemented with carbohydrates, such as fructose, glucose,galactose, lactose, and glycerol, to improve growth. Alternatively, afed batch culture is used to generate a high yield of IL-28 or IL-29polypeptide. The IL-28 or IL-29 polypeptide producing E. coli strainsare grown under conditions similar to those described for the firststage vessel used to inoculate a batch fermentation.

Following fermentation the cells are harvested by centrifugation,re-suspended in homogenization buffer and homogenized, for example, inan APV-Gaulin homogenizer (Invensys APV, Tonawanda, N.Y.) or other typeof cell disruption equipment, such as bead mills or sonicators.Alternatively, the cells are taken directly from the fermentor andhomogenized in an APV-Gaulin homogenizer. The washed inclusion body prepcan be solubilized using guanidine hydrochloride (5-8 M) or urea (7-8 M)containing a reducing agent such as beta mercaptoethanol (10-100 mM) ordithiothreitol (5-50 mM). The solutions can be prepared in Tris,phosphate, HEPES or other appropriate buffers. Inclusion bodies can alsobe solubilized with urea (2-4 M) containing sodium lauryl sulfate(0.1-2%). In the process for recovering purified IL-28 or IL-29 fromtransformed E. coli host strains in which the IL-28 or IL-29 isaccumulates as refractile inclusion bodies, the cells are disrupted andthe inclusion bodies are recovered by centrifugation. The inclusionbodies are then solubilized and denatured in 6 M guanidine hydrochloridecontaining a reducing agent. The reduced IL-28 or IL-29 is then oxidizedin a controlled renaturation step. Refolded IL-28 or IL-29 can be passedthrough a filter for clarification and removal of insoluble protein. Thesolution is then passed through a filter for clarification and removalof insoluble protein. After the IL-28 or IL-29 protein is refolded andconcentrated, the refolded IL-28 or IL-29 protein is captured in dilutebuffer on a cation exchange column and purified using hydrophobicinteraction chromatography.

Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Manassas, Va. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). See, King, L. A. and Possee, R. D., The Baculovirus ExpressionSystem: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. etal., Baculovirus Expression Vectors: A Laboratory Manual, New York,Oxford University Press, 1994; and, Richardson, C. D., Ed., BaculovirusExpression Protocols, Methods in Molecular Biology, Totowa, N.J., HumanaPress, 1995. The second method of making recombinant baculovirusutilizes a transposon-based system described by Luckow (Luckow, V. A, etal., J Virol 67:4566-79, 1993). This system is sold in the Bac-to-Backit (Life Technologies, Rockville, Md.). This system utilizes a transfervector, pFastBac1™ (Life Technologies) containing a Tn7 transposon tomove the DNA encoding the Cysteine mutant IL-28 or IL-29 polypeptideinto a baculovirus genome maintained in E. coli as a large plasmidcalled a “bacmid.” The pFastBac1™ transfer vector utilizes the AcNPVpolyhedrin promoter to drive the expression of the gene of interest, inthis case IL-28 or IL-29. However, pFastBac1™ can be modified to aconsiderable degree. The polyhedrin promoter can be removed andsubstituted with the baculovirus basic protein promoter (also known asPcor, p6.9 or MP promoter) which is expressed earlier in the baculovirusinfection, and has been shown to be advantageous for expressing secretedproteins. See, Hill-Perkins, M. S. and Possee, R. D., J. Gen. Virol.71:971-6, 1990; Bonning, B. C. et al., J. Gen. Virol. 75:1551-6, 1994;and, Chazenbalk, G. D., and Rapoport, B., J. Biol. Chem. 270:1543-9,1995. In such transfer vector constructs, a short or long version of thebasic protein promoter can be used. Moreover, transfer vectors can beconstructed which replace the native IL-28 or IL-29 secretory signalsequences with secretory signal sequences derived from insect proteins.For example, a secretory signal sequence from EcdysteroidGlucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad,Calif.), or baculovirus gp67 (PharMingen, San Diego, Calif.) can be usedin constructs to replace the native IL-28 or IL-29 secretory signalsequence. In addition, transfer vectors can include an in-frame fusionwith DNA encoding an epitope tag at the C- or N-terminus of theexpressed Cysteine mutant IL-28 or IL-29 polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci.82:7952-4, 1985). Using techniques known in the art, a transfer vectorcontaining IL-28 or IL-29 is transformed into E. Coli, and screened forbacmids which contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is isolated, using common techniques, and used to transfectSpodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus thatexpresses IL-28 or IL-29 is subsequently produced. Recombinant viralstocks are made by methods commonly used the art.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533. The use of Pichia methanolica as host for theproduction of recombinant proteins is disclosed in U.S. Pat. Nos.5,955,349, 5,888,768 and 6,001,597, U.S. Pat. No. 5,965,389, U.S. Pat.No. 5,736,383, and U.S. Pat. No. 5,854,039.

It is preferred to purify the polypeptides and proteins of the presentinvention to ≧80% purity, more preferably to ≧90% purity, even morepreferably ≧95% purity, and particularly preferred is a pharmaceuticallypure state, that is greater than 99.9% pure with respect tocontaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. Preferably, apurified polypeptide or protein is substantially free of otherpolypeptides or proteins, particularly those of animal origin.

Expressed recombinant IL-28 or IL-29 proteins (including chimericpolypeptides and multimeric proteins) are purified by conventionalprotein purification methods, typically by a combination ofchromatographic techniques. See, in general, Affinity Chromatography:Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden,1988; and Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York, 1994. Proteins comprising a polyhistidineaffinity tag (typically about 6 histidine residues) are purified byaffinity chromatography on a nickel chelate resin. See, for example,Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteins comprising aglu-glu tag can be purified by immunoaffinity chromatography accordingto conventional procedures. See, for example, Grussenmeyer et al.,supra. Maltose binding protein fusions are purified on an amylose columnaccording to methods known in the art.

IL-28 or IL-29 polypeptides can also be prepared through chemicalsynthesis according to methods known in the art, including exclusivesolid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. See, for example,Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid PhasePeptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, Ill.,1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford,1989. In vitro synthesis is particularly advantageous for thepreparation of smaller polypeptides.

Generally, the dosage of administered IL-28 or IL29 polypeptide of thepresent invention will vary depending upon such factors as the patient'sage, weight, height, sex, general medical condition and previous medicalhistory. Typically, it is desirable to provide the recipient with adosage of IL-28 or IL29 polypeptide which is in the range of from about1 pg/kg to 10 mg/kg (amount of agent/body weight of patient), although alower or higher dosage also may be administered as circumstancesdictate. One skilled in the art can readily determine such dosages, andadjustments thereto, using methods known in the art.

Administration of an IL-28 or IL29 polypeptide to a subject can betopical, inhalant, intravenous, intraarterial, intraperitoneal,intramuscular, subcutaneous, intrapleural, intrathecal, by perfusionthrough a regional catheter, or by direct intralesional injection. Whenadministering therapeutic proteins by injection, the administration maybe by continuous infusion or by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprising IL-28 orIL29 polypeptide can be prepared and inhaled with the aid of dry-powderdispersers, liquid aerosol generators, or nebulizers (e.g., Pettit andGombotz, TIBTECH 16:343 (1998); Patton et al., Adv. Drug Deliv. Rev.35:235 (1999)). This approach is illustrated by the AERX diabetesmanagement system, which is a hand-held electronic inhaler that deliversaerosolized insulin into the lungs. Studies have shown that proteins aslarge as 48,000 kDa have been delivered across skin at therapeuticconcentrations with the aid of low-frequency ultrasound, whichillustrates the feasibility of trascutaneous administration (Mitragotriet al., Science 269:850 (1995)). Transdermal delivery usingelectroporation provides another means to administer a molecule havingIL-28 or IL29 polypeptide activity (Potts et al., Pharm. Biotechnol.10:213 (1997)).

A pharmaceutical composition comprising a protein, polypeptide, orpeptide having IL-28 or IL29 polypeptide activity can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby the therapeutic proteins are combined in a mixturewith a pharmaceutically acceptable vehicle. A composition is said to bein a “pharmaceutically acceptable vehicle” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable vehicle. Other suitablevehicles are well-known to those in the art. See, for example, Gennaro(ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For purposes of therapy, molecules having IL-28 or IL29 polypeptideactivity and a pharmaceutically acceptable vehicle are administered to apatient in a therapeutically effective amount. A combination of aprotein, polypeptide, or peptide having IL-28 or IL29 polypeptideactivity and a pharmaceutically acceptable vehicle is said to beadministered in a “therapeutically effective amount” or “effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient patient. For example,an agent used to treat inflammation is physiologically significant ifits presence alleviates at least a portion of the inflammatory response.

A pharmaceutical composition comprising IL-28 or IL29 polypeptide of thepresent invention can be furnished in liquid form, in an aerosol, or insolid form. Liquid forms, are illustrated by injectable solutions,aerosols, droplets, topological solutions and oral suspensions.Exemplary solid forms include capsules, tablets, and controlled-releaseforms. The latter form is illustrated by miniosmotic pumps and implants(Bremer et al., Pharm. Biotechnol. 10:239 (1997); Ranade, “Implants inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 95-123 (CRC Press 1995); Bremer et al., “Protein Delivery withInfusion Pumps,” in Protein Delivery: Physical Systems, Sanders andHendren (eds.), pages 239-254 (Plenum Press 1997); Yewey et al.,“Delivery of Proteins from a Controlled Release Injectable Implant,” inProtein Delivery: Physical Systems, Sanders and Hendren (eds.), pages93-117 (Plenum Press 1997)). Other solid forms include creams, pastes,other topological applications, and the like.

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull. 20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

Polypeptides having IL-28 or IL29 polypeptide activity can beencapsulated within liposomes using standard techniques of proteinmicroencapsulation (see, for example, Anderson et al., Infect. Immun.31:1099 (1981), Anderson et al., Cancer Res. 50:1853 (1990), and Cohenet al., Biochim. Biophys. Acta 1063:95 (1991), Alving et al.“Preparation and Use of Liposomes in Immunological Studies,” in LiposomeTechnology, 2nd Edition, Vol. III, Gregoriadis (ed.), page 317 (CRCPress 1993), Wassef et al., Meth. Enzymol 149:124 (1987)). As notedabove, therapeutically useful liposomes may contain a variety ofcomponents. For example, liposomes may comprise lipid derivatives ofpoly(ethylene glycol) (Allen et al., Biochim. Biophys. Acta 1150:9(1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide vehicles for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises an IL-28 or IL29 polypeptide ofthe present invention. Therapeutic polypeptides can be provided in theform of an injectable solution for single or multiple doses, or as asterile powder that will be reconstituted before injection.Alternatively, such a kit can include a dry-powder disperser, liquidaerosol generator, or nebulizer for administration of a therapeuticpolypeptide. Such a kit may further comprise written information onindications and usage of the pharmaceutical composition. Moreover, suchinformation may include a statement that the IL-28 or IL29 polypeptidecomposition is contraindicated in patients with known hypersensitivityto IL-28 or IL29 polypeptide. The kit may further comprise at least oneadditional antiviral agent selected from the group of Interferon alpha,Interferon beta, Interferon gamma, Interferon omega, protease inhibitor,RNA or DNA polymerase inhibitor, nucleoside analog, antisense inhibitor,and combinations thereof. The additional antiviral agent included in thekit, for example, can be RIBAVIRIN™, PEG-INTRON®, PEGASYS®, or acombination thereof. It can also be advantageous for patients with aviral infection, such as hepatitis C, to take their medicineconsistently and get the appropriate dose for their individualizedtherapy. Thus, a kit may optionally also include a small needle, with aself-priming feature and a large, easy-to-read dosing knob. This willhelp patients feel confident that they are getting an accurate dose andoffers an easy-to-use alternative for people who may be intimidated by atraditional needle and syringe system. For example, the kit may includea disposable, one-time use precision dosing system that allows patientsto administer an IL-28 or IL-29 molecule of the present invention inthree easy steps: Mix, Dial and Deliver. (1) Mixing occurs by simplypushing down on the pen to combine the IL-28 or IL-29 molecule powderwith sterile water, both of which are stored in the body of the pen; (2)Dialing allows patients to accurately select their predeterminedindividualized dose; and (3) Delivery allows patients to inject theirindividualized dose of the medication (See, for example, ScheringPlough's PEG-INTRON REDIPEN).

IL-28 and IL-29 polypeptides of the present invention can be used intreating, ablating, curing, preventing, inhibiting, reducing, ordelaying onset of liver specific diseases, in particular liver diseasewhere viral infection is in part an etiologic agent. In particular IL-28and IL-29 polypeptides will be used to treat a mammal with a viralinfection selected from the group consisting of hepatitis A, hepatitisB, hepatitis C, and hepatitis D. When liver disease is inflammatory andcontinuing for at least six months, it is generally considered chronichepatitis. Hepatitis C virus (HCV) patients actively infected will bepositive for HCV-RNA in their blood, which is detectable by reversetranscritptase/polymerase chain reaction (RT-PCR) assays. The methods ofthe present invention will slow the progression of the liver disease.Clinically, diagnostic tests for HCV include serologic assays forantibodies and molecular tests for viral particles. Enzyme immunoassaysare available (Vrielink et al., Transfusion 37:845-849, 1997), but mayrequire confirmation using additional tests such as an immunoblot assay(Pawlotsky et al., Hepatology 27:1700-1702, 1998). Qualitative andquantitative assays generally use polymerase chain reaction techniques,and are preferred for assessing viremia and treatment response (Poynardet al., Lancet 352:1426-1432, 1998; McHutchinson et al., N. Engl. J.Med. 339:1485-1492, 1998). Several commercial tests are available, suchas, quantitative RT-PCR (Amplicor HCV Monitor™, Roche Molecular Systems,Branchburg, N.J.) and a branched DNA (deoxyribonucleic acid) signalamplification assay (Quantiplex™ HCV RNA Assay [bDNA], Chiron Corp.,Emeryville, Calif.). A non-specific laboratory test for liverinflammation or necrosis measures alanine aminotransferase level (ALT)and is inexpensive and readily available (National Institutes of HealthConsensus Development Conference Panel, Hepatology 26 (Suppl. 1):2S-10S,1997). Histologic evaluation of liver biopsy is generally considered themost accurate means for determining hepatitis progression (Yano et al.,Hepatology 23:1334-1340, 1996.) For a review of clinical tests for HCV,see, Lauer et al., N. Engl. J. Med. 345:41-52, 2001.

There are several in vivo models for testing HBV and HCV that are knownto those skilled in art. For example, the effects of IL-28 or IL-29 onmammals infected with HBV can be accessed using a woodchuck model.Briefly, woodchucks chronically infected with woodchuck hepatitis virus(WHV) develop hepatitis and hepatocellular carcinoma that is similar todisease in humans chronically infected with HBV. The model has been usedfor the preclinical assessment of antiviral activity. A chronicallyinfected WHV strain has been established and neonates are inoculatedwith serum to provide animals for studying the effects of certaincompounds using this model. (For a review, see, Tannant et al., ILAR J.42 (2):89-102, 2001). Chimpanzees may also be used to evaluate theeffect of IL-28 and IL-29 on HBV infected mammals. Using chimpanzees,characterization of HBV was made and these studies demonstrated that thechimpanzee disease was remarkably similar to the disease in humans(Barker et al., J. Infect. Dis. 132:451-458, 1975 and Tabor et al., J.Infect. Dis. 147:531-534, 1983.) The chimpanzee model has been used inevaluating vaccines (Prince et al., In: Vaccines 97, Cold Spring HarborLaboratory Press, 1997.) Therapies for HIV are routinely tested usingnon-human primates infected with simian immunodeficiency viruses (for areview, see, Hirsch et al., Adv. Pharmcol. 49:437-477, 2000 andNathanson et al., AIDS 13 (suppl. A):S113-S120, 1999.) For a review ofuse of non-human primates in HIV, hepatitis, malaria, respiratorysyncytial virus, and other diseases, see, Sibal et al., ILAR J. 42(2):74-84, 2001.

Other examples of the types of viral infections for which an IL-28 orIL-29 molecule of the present invention can be used in treating,ablating, curing, preventing, inhibiting, reducing, or delaying onset ofviral symptoms include, but are not limited to: infections caused by DNAViruses (e.g., Herpes Viruses such as Herpes Simplex viruses,Epstein-Barr virus, Cytomegalovirus; Pox viruses such as Variola (smallpox) virus; Hepadnaviruses (e.g, Hepatitis B virus); Papilloma viruses;Adenoviruses); RNA Viruses (e.g., HIV I, II; HTLV I, II; Poliovirus;Hepatitis A; Orthomyxoviruses (e.g., Influenza viruses); Paramyxoviruses(e.g., Measles virus); Rabies virus; Hepatitis C); Coronavirus (causesSevere Acute Respiratory Syndrome (SARS)); Rhinovirus, RespiratorySyncytial Virus, Norovirus, West Nile Virus, Yellow Fever, Rift VallleyVirus, Lassa Fever Virus, Ebola Virus, Lymphocytic ChoriomeningitisVirus, which replicates in tissues including liver, and the like.Moreover, examples of the types of diseases for which IL-28 and IL-29could be used include, but are not limited to: Acquiredimmunodeficiency; Hepatitis; Gastroenteritis; Hemorrhagic diseases;Enteritis; Carditis; Encephalitis; Paralysis; Brochiolitis; Upper andlower respiratory disease; Respiratory Papillomatosis; Arthritis;Disseminated disease, hepatocellular carcinoma resulting rom chronicHepatitis C infection. In addition, viral disease in other tissues maybe treated with IL-28A, IL-28B, and IL-29, for example viral meningitis,and HIV-related disease. For example, a transgenic model for testing theactivity of a therapeutic sample is described in the following examplesand described in Morrey, et al., Antiviral Ther., 3 (Suppl 3):59-68,1998.

Animal models that are used to test for efficacy in specific viruses areknown. For example, Dengue Virus can be tested using a model as such asdescribed in Huang et al., J. Gen. Virol. September; 81(Pt 9):2177-82,2000. West Nile Virus can be tested using the model as described in Xiaoet al., Emerg. Infect. Dis. July-August; 7(4):714-21, 2001 or Mashimo etal., Proc. Natl. Acad. Sci. USA. August 20; 99(17):11311-6, 2002.Venezuelan equine encephalitis virus model is described in Jackson etal., Veterinary Pathology, 28 (5): 410-418, 1991; Vogel et al., Arch.Pathol. Lab. Med. February; 120(2):164-72, 1996; Lukaszewski and Brooks,J. of Virology, 74(11):5006-5015, 2000. Rhinoviruses models aredescribed in Yin and Lomax, J. Gen. Virol. 67 (Pt 11):2335-40, 1986.Models for respiratory syncytial virus are described in Byrd and Prince,Clin. Infect. Dis. 25(6): 1363-8, 1997. Other models are known in theart and it is well within the skill of those ordinarily skilled in theart to know how to use such models.

Noroviruses (genus Norovirus, family Caliciviridae) are a group ofrelated, single-stranded RNA, nonenveloped viruses that cause acutegastroenteritis in humans. Norovirus was recently approved as theofficial genus name for the group of viruses provisionally described as“Norwalk-like viruses” (NLV). Noroviruses are estimated to cause 23million cases of acute gastroenteritis in the United States per year,and are the leading cause of gastroenteritis in the United States.

The symptoms of norovirus illness usually include nausea, vomiting,diarrhea, and some stomach cramping. Sometimes people additionally havea low-grade fever, chills, headache, muscle aches, and a general senseof tiredness. The illness often begins suddenly, and the infected personmay feel very sick. The illness is usually brief, with symptoms lastingonly about 1 or 2 days. In general, children experience more vomitingthan adults. Most people with norovirus illness have both of thesesymptoms. Currently, there is no antiviral medication that works againstnorovirus and there is no vaccine to prevent infection.

Therapeutics to Noroviruses have been difficult to identify in partbecause of a lack of good cell culture systems and animal models ofdisease. The recent identification of a murine norovirus now allowstesting of therapeutics such as IL-28 and IL-29 polypeptides of thepresent invention in a cell culture system (Wobus, Karst et al.,“Replication of Norovirus in Cell Culture Reveals a Tropism forDendritic Cells and Macrophages,” PLoS Biol, 2(12):e432, (2004)) and amouse model of disease (Karst, Wobus et al., “STAT1-dependent innateimmunity to a Norwalk-like virus,” Science, 299(5612):1575-8 (2003)).

Karst, S. M., C. E. Wobus, et al. (2003). “STAT1-dependent innateimmunity to a Norwalk-like virus.” Science, 299(5612): 1575-8.

Norwalk-like caliciviruses (Noroviruses) cause over 90% of nonbacterialepidemic gastroenteritis worldwide, but the pathogenesis of norovirusinfection is poorly understood because these viruses do not grow incultured cells and there is no small animal model. Here, we report apreviously unknown murine norovirus. Analysis of Murine Norovirus 1infection revealed that signal transducer and activator of transcription1-dependent innate immunity, but not T and B cell-dependent adaptiveimmunity, is essential for norovirus resistance. The identification ofhost molecules essential for murine norovirus resistance may providetargets for prevention or control of an important human disease.

Wobus, C. E., S. M. Karst, et al. (2004). “Replication of Norovirus inCell Culture Reveals a Tropism for Dendritic Cells and Macrophages.”PLoS Biol, 2(12): e432.

Noroviruses are understudied because these important enteric pathogenshave not been cultured to date. We found that the norovirus murinenorovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicatesin cultured primary dendritic cells and macrophages. MNV-1 growth wasinhibited by the interferon-alphabeta receptor and STAT-1, and wasassociated with extensive rearrangements of intracellular membranes. Anamino acid substitution in the capsid protein of serially passaged MNV-1was associated with virulence attenuation in vivo. This is the firstreport of replication of a norovirus in cell culture. The capacity ofMNV-1 to replicate in a STAT-1-regulated fashion and the unexpectedtropism of a norovirus for cells of the hematopoietic lineage provideimportant insights into norovirus biology.

IL-28 and IL-29 polypeptides of the present invention can be used incombination with antiviral agents, including those described above. Someof the more common treatments for viral infection include drugs thatinhibit viral replication such as ACYCLOVIR™. In addition, the combineduse of some of these agents form the basis for highly activeantiretroviral therapy (HAART) used for the treatment of AIDS. Examplesin which the combination of immunotherapy (i.e., cytokines) andantiviral drugs shows improved efficacy include the use of interferonplus RIBAVIRIN™ for the treatment of chronic hepatitis C infection(Maddrey, Semin. Liver. Dis. 19 Suppl 1:67-75, 1999) and the combineduse of IL-2 and HAART (Ross, et al, ibid.) Thus, as IL-28 and IL-29 canstimulate the immune system against disease, it can similarly be used inHAART applications.

In particular, IL-28 and IL-29 polypeptides of the present invention maybe useful in monotherapy or combination therapy with IFN-α, e.g.,PEGASYS® or PEG-INTRON® (with or without a nucleoside analog, such asRIBAVIRIN™, lamivudine, entecavir, emtricitabine, telbivudine andtenofovir) or with a nucleoside analog, such as RIBAVIRIN™, lamivudine,entecavir, emtricitabine, telbivudine and tenofovir in patients who donot respond well to IFN therapy.

These patients may not respond to IFN therapy due to having less type Iinterferon receptor on the surface of their cells (Yatsuhashi H, et al.,J Hepatol. June 30(6):995-1003, 1999; Mathai et al., J InterferonCytokine Res. September 19(9):1011-8, 1999; Fukuda et al., J Med. Virol.63(3):220-7, 2001). IL-28A, IL-28B, and IL-29 may also be useful inmonotherapy or combination therapy with IFN-α (with or without anucleoside analog, such as RIBAVIRIN™, lamivudine, entecavir,emtricitabine, and telbivudine and tenofovir) or with a nucleosideanalog, such as RIBAVIRIN™ in patients who have less type I interferonreceptor on the surface of their cells due to down-regulation of thetype I interferon receptor after type I interferon treatment (Dupont etal., J. Interferon Cytokine Res. 22(4):491-501, 2002).

IL-28 or IL-29 polypeptide may be used in combination with otherimmunotherapies including cytokines, immunoglobulin transfer, andvarious co-stimulatory molecules. In addition to antiviral drugs, IL-28and IL-29 polypeptides of the present invention can be used incombination with any other immunotherapy that is intended to stimulatethe immune system. Thus, IL-28 and IL-29 polypeptides could be used withother cytokines such as Interferon, IL-21, or IL-2. IL-28 and IL-29 canalso be added to methods of passive immunization that involveimmunoglobulin transfer, one example bring the use of antibodies totreat RSV infection in high risk patients (Meissner HC, ibid.). Inaddition, IL-28 and IL-29 polypeptides can be used with additionalco-stimulatory molecules such as 4-1BB ligand that recognize variouscell surface molecules like CD137 (Tan, J T et al., J Immunol.163:4859-68, 1999).

C. Use of IL-28A IL-28B and IL-29 in Immunocompromised Patients

IL-28 and IL-29 can be used as a monotherapy for acute and chronic viralinfections and for immunocompromised patients. Methods that enhanceimmunity can accelerate the recovery time in patients with unresolvedinfections. Immunotherapies can have an even greater impact on subsetsof immunocompromised patients such as the very young or elderly as wellas patients that suffer immunodeficiencies acquired through infection,or induced following medical interventions such as chemotherapy or bonemarrow ablation. Examples of the types of indications being treated viaimmune-modulation include; the use of IFN-α for chronic hepatitis (PerryC M, and Jarvis B, Drugs 61:2263-88, 2001), the use of IL-2 followingHIV infection (Mitsuyasu R., J. Infect. Dis. 185 Suppl 2:S115-22, 2002;and Ross R W et al., Expert Opin. Biol. Ther. 1:413-24, 2001), and theuse of IFN-α (Faro A, Springer Semin. Immunopathol. 20:425-36, 1998) fortreating Epstein Barr Virus infections following transplantation.Experiments performed in animal models indicate that IL-2 and GM-CSF mayalso be efficacious for treating EBV related diseases (Baiocchi R A etal., J Clin. Invest. 108:887-94, 2001).

IL-28 and IL-29 molecules of the present invention can be used as amonotherapy for acute and chronic viral infections and forimmunocompromised patients. Methods that enhance immunity can acceleratethe recovery time in patients with unresolved infections. In addition,IL-28 and IL-29 molecules of the present invention can be administeredto a mammal in combination with other antiviral agents such asACYCLOVIR™, RIBAVIRIN™, Interferons (e.g., PEGINTRON® and PEGASYS®),Serine Protease Inhibitors, Polymerase Inhibitors, Nucleoside Analogs,Antisense Inhibitors, and combinations thereof, to treat, ablate, cure,prevent, inhibit, reduce, or delay the onset of a viral infectionselected from the group of hepatitis A, hepatitis B, hepatitis C,hepatitis D, respiratory syncytial virus, herpes virus, Epstein-Barrvirus, influenza virus, adenovirus, parainfluenza virus, Severe AcuteRespiratory Syndrome, rhino virus, coxsackie virus, vaccinia virus, westnile virus, dengue virus, venezuelan equine encephalitis virus, pichindevirus, and polio virus. IL-28 and IL-29 polypeptides of the presentinvention can also be used in combination with other immunotherapiesincluding cytokines, immunoglobulin transfer, and various co-stimulatorymolecules. In addition, IL-28 and IL-29 molecules of the presentinvention can be used to treat a mammal with a chronic or acute viralinfection that has resulted liver inflammation, thereby reducing theviral infection and/or liver inflammation. In particular IL-28 and IL-29will be used to treat a mammal with a viral infection selected from thegroup of hepatitis A, hepatitis B, hepatitis C, and/or hepatitis D.IL-28 and IL-29 molecules of the present invention can also be used asan antiviral agent in viral infections selected from the groupconsisting of respiratory syncytial virus, herpes virus, Epstein-Barrvirus, influenza virus, adenovirus, parainfluenza virus, Severe AcuteRespiratory Syndrome, rhino virus, coxsackie virus, vaccinia virus, westnile virus, dengue virus, venezuelan equine encephalitis virus, pichindevirus and polio virus.

The present invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Induction of IL-28A IL-29 and IL-28B by poly I:C andviral infection

Freshly isolated human peripheral blood mononuclear cells were grown inthe presence of polyinosinic acid-polycytidylic acid (poly I:C; 100□g/ml) (SIGMA; St. Louis, Mo.), encephalomyocarditis virus (EMCV) withan MOI of 0.1, or in medium alone. After a 15 h incubation, total RNAwas isolated from cells and treated with RNase-free DNase. 100 ng totalRNA was used as template for one-step RT-PCR using the SuperscriptOne-Step RT-PCR with Platinum Taq kit and gene-specific primers assuggested by the manufacturer (Invitrogen).

Low to undetectable amounts of human IL-28A, IL-28B, and IL-29, IFN-□and IFN-□ RNA were seen in untreated cells. In contrast, the amount ofIL-28A, IL-29, IL-28B RNA was increased by both poly I:C treatment andviral infection, as was also seen for the type I interferons. Theseexperiments indicate that IL-28A, IL-29, IL-28B, like type Iinterferons, can be induced by double-stranded RNA or viral infection.

Example 2 IL-28 and IL-29 Signaling Activity Compared to IFNα in HepG2Cells A. Cell Transfections

HepG2 cells were transfected as follows: 700,000 HepG2 cells/well (6well plates) were plated approximately 18 h prior to transfection in 2milliliters DMEM+10% fetal bovine serum. Per well, 1 microgrampISRE-Luciferase DNA (Stratagene) and 1 microgram pIRES2-EGFP DNA(Clontech) were added to 6 microliters Fugene 6 reagent (RocheBiochemicals) in a total of 100 microliters DMEM. This transfection mixwas added 30 minutes later to the pre-plated HepG2 cells. Twenty-fourhours later the transfected cells were removed from the plate usingtrypsin-EDTA and replated at approximately 25,000 cells/well in 96 wellmicrotiter plates. Approximately 18 h prior to ligand stimulation, mediawas changed to DMEM+0.5% FBS.

B. Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37° C. in DMEM+0.5% FBS, transfected cells werestimulated with 100 ng/ml IL-28A, IL-29, IL-28B, zcyto24, zcyto25 andhuIFN-α2a ligands. Following a 4-hour incubation at 37° degrees, thecells were lysed, and the relative light units (RLU) were measured on aluminometer after addition of a luciferase substrate. The resultsobtained are shown as the fold induction of the RLU of the experimentalsamples over the medium alone control (RLU of experimental samples/RLUof medium alone=fold induction). Table 5 shows that IL-28A, IL-29,IL-28B, zcyto24 and zcyto25 induce ISRE signaling in human HepG2 livercells transfected with ISRE-luciferase.

TABLE 5 Fold Induction of Cytokine-dependent ISRE Signaling in HepG2Cells Cytokine Fold Induction IL-28A 5.6 IL-29 4 IL-28B 5.8 Zcyto24 4.7Zcyto25 3 HuIFN-a2a 5.8

Example 3 IL-29 Antiviral Activity Compared to IFNα in HepG2 Cells

An antiviral assay was adapted for EMCV (American Type CultureCollection # VR-129B, Manassas, Va.) with human cells (Familletti, P.,et al., Methods Enzym. 78: 387-394, 1981). Cells were plated withcytokines and incubated 24 hours prior to challenge by EMCV at amultiplicity of infection of 0.1 to 1. The cells were analyzed forviability with a dye-uptake bioassay 24 hours after infection (Berg, K.,et al., Apmis 98: 156-162, 1990). Target cells were given MTT andincubated at 37° C. for 2 hours. A solubiliser solution was added,incubated overnight at 37° C. and the optical density at 570 nm wasdetermined. OD570 is directly proportional to antiviral activity.

The results show the antiviral activity when IL-29 and IFN on weretested with HepG2 cells: IL-29, IFÑ□ and IF

α-2a were added at varying concentration to HepG2 cells prior to EMCVinfection and dye-uptake assay. The mean and standard deviation of theOD570 from triplicate wells is plotted. OD570 is directly proportionalto antiviral activity. For IL-29, the EC50 was 0.60 ng/ml; for IFN-α2a,the EC50 was 0.57 ng/ml; and for IFN-β, the EC50 was 0.46 ng/ml.

Example 4 IL-28RA mRNA Expression in Liver and Lymphocyte Subsets

In order to further examine the mRNA distribution for IL-28RA,semi-quantitative RT-PCR was performed using the SDS 7900HT system(Applied Biosystems, CA). One-step RT-PCR was performed using 100 ngtotal RNA for each sample and gene-specific primers. A standard curvewas generated for each primer set using Bjab RNA and all sample valueswere normalized to HPRT. The normalized results are summarized in Tables6-8. The normalized values for IFNAR2 and CRF2-4 are also shown.

Table 6: B and T cells express significant levels of IL-28RA mRNA. Lowlevels are seen in dendritic cells and most monocytes.

TABLE 6 Cell/Tissue IL-28RA IFNAR2 CRF2-4 Dendritic Cells unstim .04 5.99.8 Dendritic Cells +IFNg .07 3.6 4.3 Dendritic Cells .16 7.85 3.9 CD14+stim'd with LPS/IFNg .13 12 27 CD14+ monocytes resting .12 11 15.4 HuCD14+ Unact. 4.2 TBD TBD Hu CD14+ 1 ug/ml LPS act. 2.3 TBD TBD H.Inflamed tonsil 3 12.4 9.5 H. B-cells + PMA/Iono 4 & 24 hrs 3.6 1.3 1.4Hu CD19+ resting 6.2 TBD TBD Hu CD19+ 4 hr. PMA/Iono 10.6 TBD TBD HuCD19+ 24 hr Act. PMA/Iono 3.7 TBD TBD IgD+ B-cells 6.47 13.15 6.42 IgM+B-cells 9.06 15.4 2.18 IgD− B-cells 5.66 2.86 6.76 NKCells + PMA/Iono 06.7 2.9 Hu CD3+ Unactivated 2.1 TBD TBD CD4+ resting .9 8.5 29.1 CD4+Unstim 18 hrs 1.6 8.4 13.2 CD4+ + Poly I/C 2.2 4.5 5.1 CD4+ + PMA/Iono.3 1.8 .9 CD3 neg resting 1.6 7.3 46 CD3 neg unstim 18 hrs 2.4 13.2 16.8CD3 neg + Poly I/C 18 hrs 5.7 7 30.2 CD3 neg + LPS 18 hrs 3.1 11.9 28.2CD8+ unstim 18 hrs 1.8 4.9 13.1 CD8+ stim'd with PMA/Ion 18 hrs .3 .61.1

As shown in Table 7, normal liver tissue and liver derived cell linesdisplay substantial levels of IL-28RA and CRF2-4 mRNA.

TABLE 7 Cell/Tissue IL-28RA IFNAR2 CRF2-4 HepG2 1.6 3.56 2.1 HepG2 UGAR5/10/02 1.1 1.2 2.7 HepG2, CGAT HKES081501C 4.3 2.1 6 HuH7 5/10/02 1.6316 2 HuH7 hepatoma - CGAT 4.2 7.2 3.1 Liver, normal - CGAT 11.7 3.2 8.4#HXYZ020801K Liver, NAT - Normal adjacent tissue 4.5 4.9 7.7 Liver,NAT - Normal adjacent tissue 2.2 6.3 10.4 Hep SMVC hep vein 0 1.4 6.5Hep SMCA hep. Artery 0 2.1 7.5 Hep. Fibro 0 2.9 6.2 Hep. Ca. 3.8 2.9 5.8Adenoca liver 8.3 4.2 10.5 SK-Hep-1 adenoca. Liver .1 1.3 2.5 AsPC-1 Hu.Pancreatic adenocarc. .7 .8 1.3 Hu. Hep. Stellate cells .025 4.4 9.7

As shown in Table 8, primary airway epithelial cells contain abundantlevels of IL-28RA and CRF2-4.

TABLE 8 Cell/Tissue IL-28RA IFNAR2 CRF2-4 U87MG - glioma 0  .66  .99NHBE unstim 1.9 1.7 8.8 NHBE + TNF-alpha 2.2 5.7 4.6 NHBE + poly I/C 1.8nd nd Small Airway Epithelial Cells 3.9 3.3 27.8  NHLF - Normal humanlung fibroblasts 0 nd nd

As shown in Table 8, IL-28RA is present in normal and diseased liverspecimens, with increased expression in tissue from Hepatitis C andHepatitis B infected specimens.

TABLE 8 Cell/Tissue IL-28RA CRF2-4 IFNAR2 Liver with CoagulationNecrosis 8.87 15.12 1.72 Liver with Autoimmune Hepatitis 6.46 8.90 3.07Neonatal Hepatitis 6.29 12.46 6.16 Endstage Liver disease 4.79 17.0510.58 Fulminant Liver Failure 1.90 14.20 7.69 Fulminant Liver failure2.52 11.25 8.84 Cirrhosis, primary biliary 4.64 12.03 3.62 CirrhosisAlcoholic (Laennec's) 4.17 8.30 4.14 Cirrhosis, Cryptogenic 4.84 7.135.06 Hepatitis C+, with cirrhosis 3.64 7.99 6.62 Hepatitis C+ 6.32 11.297.43 Fulminant hepatitis secondary to Hep A 8.94 21.63 8.48 Hepatitis C+7.69 15.88 8.05 Hepatitis B+ 1.61 12.79 6.93 Normal Liver 8.76 5.42 3.78Normal Liver 1.46 4.13 4.83 Liver NAT 3.61 5.43 6.42 Liver NAT 1.9710.37 6.31 Hu Fetal Liver 1.07 4.87 3.98 Hepatocellular Carcinoma 3.583.80 3.22 Adenocarcinoma Liver 8.30 10.48 4.17 hep. SMVC, hep. Vein 0.006.46 1.45 Hep SMCA hep. Artery 0.00 7.55 2.10 Hep. Fibroblast 0.00 6.202.94 HuH7 hepatoma 4.20 3.05 7.24 HepG2 Hepatocellular carcinoma 3.405.98 2.11 SK-Hep-1 adenocar. Liver 0.03 2.53 1.30 HepG2 Unstim 2.06 2.982.28 HepG2 + zcyto21 2.28 3.01 2.53 HepG2 + IFNα 2.61 3.05 3.00 NormalFemale Liver - degraded 1.38 6.45 4.57 Normal Liver - degraded 1.93 4.996.25 Normal Liver - degraded 2.41 2.32 2.75 Disease Liver - degraded2.33 3.00 6.04 Primary Hepatocytes from Clonetics 9.13 7.97 13.30

As shown in Tables 9-13, IL-28RA is detectable in normal B cells, Blymphoma cell lines, T cells, T lymphoma cell lines (Jurkat), normal andtransformed lymphocytes (B cells and T cells) and normal humanmonocytes.

TABLE 9 HPRT IL-28RA IL-28RA IFNR2 CRF2-4 Mean Mean norm IFNAR2 normCRF2-4 Norm CD14+ 24 hr unstim #A38 13.1 68.9 5.2 92.3 7.0 199.8 15.2CD14+ 24 hr stim #A38 6.9 7.6 1.1 219.5 31.8 276.6 40.1 CD14+ 24 hrunstim #A112 17.5 40.6 2.3 163.8 9.4 239.7 13.7 CD14+ 24 hr stim #A11211.8 6.4 0.5 264.6 22.4 266.9 22.6 CD14+ rest #X 32.0 164.2 5.1 1279.739.9 699.9 21.8 CD14+ + LPS #X 21.4 40.8 1.9 338.2 15.8 518.0 24.2 CD14+24 hr unstim #A39 26.3 86.8 3.3 297.4 11.3 480.6 18.3 CD14+ 24 hr stim#A39 16.6 12.5 0.8 210.0 12.7 406.4 24.5 HL60 Resting 161.2 0.2 0.0214.2 1.3 264.0 1.6 HL60 + PMA 23.6 2.8 0.1 372.5 15.8 397.5 16.8 U937Resting 246.7 0.0 0.0 449.4 1.8 362.5 1.5 U937 + PMA 222.7 0.0 0.0 379.21.7 475.9 2.1 Jurkat Resting 241.7 103.0 0.4 327.7 1.4 36.1 0.1 JurkatActivated 130.7 143.2 1.1 Colo205 88.8 43.5 0.5 HT-29 26.5 30.5 1.2

TABLE 10 HPRT SD IL-28RA SD Mono 24 hr unstim #A38 0.6 2.4 Mono 24 hrstim #A38 0.7 0.2 Mono 24 hr unstim 2.0 0.7 #A112 Mono 24 hr stim #A1120.3 0.1 Mono rest #X 5.7 2.2 Mono + LPS #X 0.5 1.0 Mono 24 hr unstim#A39 0.7 0.8 Mono 24 hr stim #A39 0.1 0.7 HL60 Resting 19.7 0.1 HL60 +PMA 0.7 0.4 U937 Resting 7.4 0.0 U937 + PMA 7.1 0.0 Jurkat Resting 3.71.1 Jurkat Activated 2.4 1.8 Colo205 1.9 0.7 HT-29 2.3 1.7

TABLE 11 Mean Mean Mean IL- Mean Hprt IFNAR2 28RA CRF CD3+/CD4+ 0 10.185.9 9.0 294.6 CD4/CD3+ Unstim 18 hrs 12.9 108.7 20.3 170.4 CD4+/CD3+ +Poly I/C 18 hrs 24.1 108.5 52.1 121.8 CD4+/CD3+ + PMA/Iono 18 hrs 47.883.7 16.5 40.8 CD3 neg 0 15.4 111.7 24.8 706.1 CD3 neg unstim 18 hrs15.7 206.6 37.5 263.0 CD3 neg + Poly I/C 18 hrs 9.6 67.0 54.7 289.5 CD3neg + LPS 18 hrs 14.5 173.2 44.6 409.3 CD8+ Unstim. 18 hrs 6.1 29.7 11.179.9 CD8+ + PMA/Iono 18 hrs 78.4 47.6 26.1 85.5 12.8.1 - NHBE Unstim47.4 81.1 76.5 415.6 12.8.2 - NHBE + TNF-alpha 42.3 238.8 127.7 193.9SAEC 15.3 49.9 63.6 426.0

TABLE 12 IL-28RA CRF IFNAR2 IL-28RA CRF IFNAR2 Norm Norm Norm SD SD SDCD3+/CD4+ 0 0.9 29.1 8.5 0.1 1.6 0.4 CD4/CD3+ Unstim 18 hrs 1.6 13.2 8.40.2 1.6 1.4 CD4+/CD3+ + Poly I/C 18 hrs 2.2 5.1 4.5 0.1 0.3 0.5CD4+/CD3+ + PMA/Iono 18 hrs 0.3 0.9 1.8 0.0 0.1 0.3 CD3 neg 0 1.6 46.07.3 0.2 4.7 1.3 CD3 neg unstim 18 hrs 2.4 16.8 13.2 0.4 2.7 2.3 CD3neg + Poly I/C 18 hrs 5.7 30.2 7.0 0.3 1.7 0.8 CD3 neg + LPS 18 hrs 3.128.2 11.9 0.4 5.4 2.9 CD8+ Unstim. 18 hrs 1.8 13.1 4.9 0.1 1.1 0.3CD8+ + PMA/Iono 18 hrs 0.3 1.1 0.6 0.0 0.1 0.0 12.8.1 - NHBE Unstim 1.68.8 1.7 0.1 0.4 0.1 12.8.2 - NHBE + TNF-alpha 3.0 4.6 5.7 0.1 0.1 0.1SAEC 4.1 27.8 3.3 0.2 1.1 0.3

TABLE 13 SD SD IL- SD SD Hprt IFNAR2 28RA CRF CD3+/CD4+ 0 0.3 3.5 0.612.8 CD4/CD3+ Unstim 18 hrs 1.4 13.7 1.1 8.5 CD4+/CD3+ + Poly I/C 18 hrs1.3 9.8 1.6 3.4 CD4+/CD3+ + PMA/Iono 18 hrs 4.0 10.3 0.7 3.7 CD3 neg 01.4 16.6 1.6 28.6 CD3 neg unstim 18 hrs 2.4 16.2 2.7 12.6 CD3 neg + PolyI/C 18 hrs 0.5 7.0 1.0 8.3 CD3 neg + LPS 18 hrs 1.0 39.8 5.6 73.6 CD8+Unstim. 18 hrs 0.2 1.6 0.5 6.1 CD8+ + PMA/Iono 18 hrs 1.3 1.7 0.2 8.112.8.1 - NHBE Unstim 2.4 5.6 2.7 2.8 12.8.2 - NHBE + TNF-alpha 0.5 3.43.5 3.4 SAEC 0.5 4.8 1.8 9.9

Example 5 Mouse IL-28 does not Effect Daudi Cell Proliferation

Human Daudi cells were suspended in RPMI+10% FBS at 50,000cells/milliliter and 5000 cells were plated per well in a 96 well plate.IL-29-CEE (IL-29 conjugated with glu tag), IFN-□ or IFN-□2a was added in2-fold serial dilutions to each well. IL-29-CEE was used at aconcentration range of from 1000 ng/ml to 0.5 ng/ml. IFN-□ was used at aconcentration range from 125 ng/ml to 0.06 ng/ml. IFN-□2a was used at aconcentration range of from 62 ng/ml to 0.03 ng/ml. Cells were incubatedfor 72 h at 37° C. After 72 hours Alamar Blue (Accumed, Chicago, Ill.)was added at 20 microliters/well. Plates were further incubated at 37°C., 5% CO, for 24 hours. Plates were read on the Fmax™ plate reader(Molecular Devices, Sunnyvale, Calif.) using the SoftMax™ Pro program,at wavelengths 544 (Excitation) and 590 (Emission). Alamar Blue gives afluourometric readout based on the metabolic activity of cells, and isthus a direct measurement of cell proliferation in comparison to anegative control. The results indicate that IL-29-CEE, in contrast toIFN-□2a, has no significant effect on proliferation of Daudi cells.

Example 6 Mouse IL-28 Does not have Antiproliferative Effect on Mouse BCells

Mouse B cells were isolated from 2 Balb/C spleens (7 months old) bydepleting CD43+ cells using MACS magnetic beads. Purified B cells werecultured in vitro with LPS, anti-IgM or anti-CD40 monoclonal antibodies.Mouse IL-28 or mouse IFNα was added to the cultures and ³H-thymidine wasadded at 48 hrs. and ³H-thymidine incorporation was measured after 72hrs. culture.

IFNα at 10 ng/ml inhibited ³H-thymidine incorporation by mouse B cellsstimulated with either LPS or anti-IgM. However mouse IL-28 did notinhibit ³H-thymidine incorporation at any concentration tested including1000 ng/ml. In contrast, both mIFNα and mouse IL-28 increased ³Hthymidine incorporation by mouse B cells stimulated with anti-CD40 MAb.

These data demonstrate that mouse IL-28 unlike IFNa displays noantiproliferative activity even at high concentrations. In addition,zcyto24 enhances proliferation in the presence of anti-CD40 MAbs. Theresults illustrate that mouse IL-28 differs from IFNα in that mouseIL-28 does not display antiproliferative activity on mouse B cells, evenat high concentrations. In addition, mouse IL-28 enhances proliferationin the presence of anti-CD40 monoclonal antibodies.

Example 7 Bone Marrow Expansion Assay

Fresh human marrow mononuclear cells (Poietic Technologies,Gaithersburg, Md.) were adhered to plastic for 2 hrs in □MEM, 10% FBS,50 micromolar {tilde over (□)}mercaptoethanol, 2 ng/ml FLT3L at 37° C.Non adherent cells were then plated at 25,000 to 45,000 cells/well (96well tissue culture plates) in □MEM, 10% FBS, 50 micromolar {tilde over(□)}mercaptoethanol, 2 ng/ml FLT3L in the presence or absence of 1000ng/ml IL-29-CEE, 100 ng/ml IL-29-CEE, 10 ng/ml IL-29-CEE, 100 ng/mlIFN-□2a, 10 ng/ml IF

-□2a or 1 ng/ml IF

-□2a. These cells were incubated with a variety of cytokines to test forexpansion or differentiation of hematopoietic cells from the marrow (20ng/ml IL-2, 2 ng/ml IL-3, 20 ng/ml IL-4, 20 ng/ml IL-5, 20 ng/ml IL-7,20 ng/ml IL-10, 20 ng/ml IL-12, 20 ng/ml IL-15, 10 ng/ml IL-21 or noadded cytokine). After 8 to 12 days Alamar Blue (Accumed, Chicago, Ill.)was added at 20 microliters/well. Plates were further incubated at 37°C., 5% CO, for 24 hours. Plates were read on the Fmax™ plate reader(Molecular Devices Sunnyvale, Calif.) using the SoftMax™ Pro program, atwavelengths 544 (Excitation) and 590 (Emission). Alamar Blue gives afluourometric readout based on the metabolic activity of cells, and isthus a direct measurement of cell proliferation in comparison to anegative control.

IF

-□2a caused a significant inhibition of bone marrow expansion under allconditions tested. In contrast, IL-29 had no significant effect onexpansion of bone marrow cells in the presence of IL-3, IL-4, IL-5,IL-7, IL-10, IL-12, IL-21 or no added cytokine. A small inhibition ofbone marrow cell expansion was seen in the presence of IL-2 or IL-15.

Example 8 Inhibition of IL-28 and IL-29 Signaling with Soluble Receptor(zcytoR19/CRF2-4) A. Signal Transduction Reporter Assay

A signal transduction reporter assay can be used to show the inhibitorproperties of zcytor19-Fc4 homodimeric and zcytor19-Fc/CRF2-4-Fcheterodimeric soluble receptors on zcyto20, zcyto21 and zcyto24signaling. Human embryonal kidney (HEK) cells overexpressing thezcytor19 receptor are transfected with a reporter plasmid containing aninterferon-stimulated response element (ISRE) driving transcription of aluciferase reporter gene. Luciferase activity following stimulation oftransfected cells with ligands (including zcyto20 (SEQ ID NO:18),zcyto21 (SEQ ID NO:20), zcyto24 (SEQ ID NO: 8)) reflects the interactionof the ligand with soluble receptor.

B. Cell Transfections

HEK cells overexpressing zcytor19 were transfected as follows: 700,000293 cells/well (6 well plates) were plated approximately 18 h prior totransfection in 2 milliliters DMEM+10% fetal bovine serum. Per well, 1microgram pISRE-Luciferase DNA (Stratagene) and 1 microgram pIRES2-EGFPDNA (Clontech) were added to 6 microliters Fugene 6 reagent (RocheBiochemicals) in a total of 100 microliters DMEM. This transfection mixwas added 30 minutes later to the pre-plated 293 cells. Twenty-fourhours later the transfected cells were removed from the plate usingtrypsin-EDTA and replated at approximately 25,000 cells/well in 96 wellmicrotiter plates. Approximately 18 h prior to ligand stimulation, mediawas changed to DMEM+0.5% FBS.

C. Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37° C. in DMEM+0.5% FBS, transfected cells werestimulated with 10 ng/ml zcyto20, zcyto21 or zcyto24 and 10micrograms/ml of the following soluble receptors; human zcytor19-Fchomodimer, human zcytor19-Fc/human CRF2-4-Fc heterodimer, humanCRF2-4-Fc homodimer, murine zcytor19-Ig homodimer. Following a 4-hourincubation at 37° C., the cells were lysed, and the relative light units(RLU) were measured on a luminometer after addition of a luciferasesubstrate. The results obtained are shown as the percent inhibition ofligand-induced signaling in the presence of soluble receptor relative tothe signaling in the presence of PBS alone. Table 13 shows that thehuman zcytor19-Fc/human CRF2-4 heterodimeric soluble receptor is able toinhibit zcyto20, zcyto21 and zcyto24-induced signaling between 16 and45% of control. The human zcytor19-Fc homodimeric soluble receptor isalso able to inhibit zcyto21-induced signaling by 45%. No significanteffects were seen with huCRF2-4-Fc or muzcytor19-Ig homodimeric solublereceptors.

TABLE 14 Percent Inhibition of Ligand-induced Interferon StimulatedResponse Element (ISRE) Signaling by Soluble Receptors Huzcytor19-HuCRF2- Ligand Fc/huCRF2-4-Fc Huzcytor19-Fc 4-Fc Muzcytor19-Ig Zcyto2016% 92% 80% 91% Zcyto21 16% 45% 79% 103% Zcyto24 47% 90% 82% 89%

Example 9 IL-28 and IL-29 Inhibit HIV Replication in Fresh Human PBMCs

Human immunodeficiency virus (HIV) is a pathogenic retrovirus thatinfects cells of the immune system. CD4 T cells and monocytes are theprimary infected cell types. To test the ability of IL-28 and IL-29 toinhibit HIV replication in vitro, PBMCs from normal donors were infectedwith the HIV virus in the presence of IL-28, IL-29 andMetIL-29C172S-PEG.

Fresh human peripheral blood mononuclear cells (PBMCs) were isolatedfrom whole blood obtained from screened donors who were seronegative forHIV and HBV. Peripheral blood cells were pelleted/washed 2-3 times bylow speed centrifugation and resuspended in PBS to remove contaminatingplatelets. The washed blood cells were diluted 1:1 with Dulbecco'sphosphate buffered saline (D-PBS) and layered over 14 mL of LymphocyteSeparation Medium ((LSM; Cellgro™ by Mediatech, Inc. Herndon, Va.);density 1.078+/−0.002 g/ml) in a 50 mL centrifuge tube and centrifugedfor 30 minutes at 600×G. Banded PBMCs were gently aspirated from theresulting interface and subsequently washed 2× in PBS by low speedcentrifugation. After the final wash, cells were counted by trypan blueexclusion and resuspended at 1×10⁷ cells/mL in RPMI 1640 supplementedwith 15% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 4 μg/mL PHA-P. Thecells were allowed to incubate for 48-72 hours at 37° C. Afterincubation, PBMCs were centrifuged and resuspended in RPMI 1640 with 15%FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10μg/mL gentamycin, and 20 U/mL recombinant human IL-2. PBMCs weremaintained in the medium at a concentration of 1-2×10⁶ cells/mL withbiweekly medium changes until used in the assay protocol. Monocytes weredepleted from the culture as the result of adherence to the tissueculture flask.

For the standard PBMC assay, PHA-P stimulated cells from at least twonormal donors were pooled, diluted in fresh medium to a finalconcentration of 1×10⁶ cells/mL, and plated in the interior wells of a96 well round bottom microplate at 50 μL/well (5×10⁴ cells/well). Testdilutions were prepared at a 2× concentration in microtiter tubes and100 μL of each concentration was placed in appropriate wells in astandard format. IL-28, IL-29 and MetIL-29C172S-PEG were added atconcentrations from 0-10 μg/ml, usually in ½ log dilutions. 50 μL of apredetermined dilution of virus stock was placed in each test well(final MOI of 0.1). Wells with only cells and virus added were used forvirus control. Separate plates were prepared identically without virusfor drug cytotoxicity studies using an MTS assay system. The PBMCcultures were maintained for seven days following infection, at whichtime cell-free supernatant samples were collected and assayed forreverse transcriptase activity and p24 antigen levels.

A decrease in reverse transcriptase activity or p24 antigen levels withIL-28, IL-29 and MetIL-29C172S-PEG would be indicators of antiviralactivity. Result would demonstrate that IL-28 and IL-29 may havetherapeutic value in treating HIV and AIDS.

Example 10 IL-28 and IL-29 Inhibit GBV-B Replication in Marmoset LiverCells

HCV is a member of the Flaviviridae family of RNA viruses. HCV does notreplicate well in either ex-vivo or in vitro cultures and therefore,there are no satisfactory systems to test the anti-HCV activity ofmolecules in vitro. GB virus B (GBV-B) is an attractive surrogate modelfor use in the development of anti-HCV antiviral agents since it has arelatively high level of sequence identity with HCV and is ahepatotropic virus. To date, the virus can only be grown in the primaryhepatocytes of certain non-human primates. This is accomplished byeither isolating hepatocytes in vitro and infecting them with GBV-B, orby isolating hepatocytes from GBV-B infected marmosets and directlyusing them with antiviral compounds.

The effects of IL-28, IL-29 and MetIL-29C172S-PEG are assayed on GBV-Bextracellular RNA production by TaqMan RT-PCR and on cytotoxicity usingCellTiter96® reagent (Promega, Madison, Wis.) at six half-log dilutionsIL-28, IL-29 or MetIL-29C172S-PEG polypeptide in triplicate. Untreatedcultures serve as the cell and virus controls. Both RIBAVIRIN® (200μg/ml at the highest test concentration) and IFN-α (5000 IU/ml at thehighest test) are included as positive control compounds. Primaryhepatocyte cultures are isolated and plated out on collagen-coatedplates. The next day the cultures are treated with the test samples(IL-28, IL-29, MetIL-29C172S-PEG, IFNα, or RIBAVIRIN®) for 24 hr beforebeing exposed to GBV-B virions or treated directly with test sampleswhen using in vivo infected hepatocytes. Test samples and media areadded the next day, and replaced three days later. Three to four dayslater (at day 6-7 post test sample addition) the supernatant iscollected and the cell numbers quantitated with CellTiter96®. Viral RNAis extracted from the supernatant and quantified with triplicatereplicates in a quantitative TaqMan RT-PCR assay using an in vitrotranscribed RNA containing the RT-PCR target as a standard. The averageof replicate samples is computed. Inhibition of virus production isassessed by plotting the average RNA and cell number values of thetriplicate samples relative to the untreated virus and cell controls.The inhibitory concentration of drug resulting in 50% inhibition ofGBV-B RNA production (IC50) and the toxic concentration resulting indestruction of 50% of cell numbers relative to control values (TC50) arecalculated by interpolation from graphs created with the data.

Inhibition of the GBV-B RNA production by IL-28 and 29 is an indicationof the antiviral properties of IL-28 and IL-29 on this Hepatitis C-likevirus on hepatocytes, the primary organ of infection of Hepatitis C, andpositive results suggest that IL-28 or IL-29 may be useful in treatingHCV infections in humans.

Example 11 IL-28, IL-29 and MetIL-29C172S-PEG Inhibit HBV Replication inWT10 Cells

Chronic hepatitis B (HBV) is one of the most common and severe viralinfections of humans belonging to the Hepadnaviridae family of viruses.To test the antiviral activities of IL-28 and IL-29 against HBV, IL-28,IL-29 and MetIL-29C172S-PEG were tested against HBV in an in vitroinfection system using a variant of the human liver line HepG2. IL-28,IL-29 and MetIL-29C172S-PEG inhibited viral replication in this system,suggesting therapeutic value in treating HBV in humans.

WT10 cells are a derivative of the human liver cell line HepG2 2.2.15.WT10 cells are stably transfected with the HBV genome, enabling stableexpression of HBV transcripts in the cell line (Fu and Cheng,Antimicrobial Agents Chemother. 44(12):3402-3407, 2000). In the WT10assay the drug in question and a 3TC control will be assayed at fiveconcentrations each, diluted in a half-log series. The endpoints areTaqMan PCR for extracellular HBV DNA (IC50) and cell numbers usingCellTiter96 reagent (TC50). The assay is similar to that described byKorba et al. Antiviral Res. 15(3):217-228, 1991 and Korba et al.,Antiviral Res. 19(1):55-70, 1992. Briefly, WT10 cells are plated in96-well microtiter plates. After 16-24 hours the confluent monolayer ofHepG2-2.2.15 cells is washed and the medium is replaced with completemedium containing varying concentrations of a test samples intriplicate. 3TC is used as the positive control, while media alone isadded to cells as a negative control (virus control, VC). Three dayslater the culture medium is replaced with fresh medium containing theappropriately diluted test samples. Six days following the initialaddition of the test compound, the cell culture supernatant iscollected, treated with pronase and DNAse, and used in a real-timequantitative TaqMan PCR assay. The PCR-amplified HBV DNA is detected inreal-time by monitoring increases in fluorescence signals that resultfrom the exonucleolytic degradation of a quenched fluorescent probemolecule that hybridizes to the amplified HBV DNA. For each PCRamplification, a standard curve is simultaneously generated usingdilutions of purified HBV DNA. Antiviral activity is calculated from thereduction in HBV DNA levels (IC₅₀). A dye uptake assay is then employedto measure cell viability which is used to calculate toxicity (TC₅₀).The therapeutic index (TI) is calculated as TC₅₀/IC₅₀.

IL-28, IL-29 and MetIL-29C172S-PEG inhibited HepB viral replication inWT10 cells with an IC50<0.032 ug/ml. This demonstrates antiviralactivity of IL-28 and IL-29 against HBV grown in liver cell lines,providing evidence of therapeutic value for treating HBV in humanpatients.

Example 12 IL-28, IL-29 and MetIL-29C172S-PEG Inhibit BVDV Replicationin Bovine Kidney Cells

HCV is a member of the Flaviviridae family of RNA viruses. Other virusesbelonging to this family are the bovine viral diarrhea virus (BVDV) andyellow fever virus (YFV). HCV does not replicate well in either ex vivoor in vitro cultures and therefore there are no systems to test anti-HCVactivity in vitro. The BVDV and YFV assays are used as surrogate virusesfor HCV to test the antiviral activities against the Flavivirida familyof viruses.

The antiviral effects of IL-28, IL-29 and MetIL-29C172S-PEG wereassessed in inhibition of cytopathic effect assays (CPE). The assaymeasured cell death using Madin-Darby bovine kidney cells (MDBK) afterinfection with cytopathic BVDV virus and the inhibition of cell death byaddition of IL-28, IL-29 and MetIL-29C172S-PEG. The MDBK cells werepropagated in Dulbecco's modified essential medium (DMEM) containingphenol red with 10% horse serum, 1% glutamine and 1%penicillin-streptomycin. CPE inhibition assays were performed in DMEMwithout phenol red with 2% FBS, 1% glutamine and 1% Pen-Strep. On theday preceding the assays, cells were trypsinized (1% trypsin-EDTA),washed, counted and plated out at 10⁴ cells/well in a 96-wellflat-bottom BioCoat® plates (Fisher Scientific, Pittsburgh, Pa.) in avolume of 100 μl/well. The next day, the medium was removed and apre-titered aliquot of virus was added to the cells. The amount of viruswas the maximum dilution that would yield complete cell killing (>80%)at the time of maximal CPE development (day 7 for BVDV). Cell viabilitywas determined using a CellTiter96® reagent (Promega) according to themanufacturer's protocol, using a Vmax plate reader (Molecular Devices,Sunnyvale, Calif.). Test samples were tested at six concentrations each,diluted in assay medium in a half-log series. IFNα and RIBAVIRIN® wereused as positive controls. Test sample were added at the time of viralinfection. The average background and sample color-corrected data forpercent CPE reduction and percent cell viability at each concentrationwere determined relative to controls and the IC₅₀ calculated relative tothe TC₅₀.

IL-28, IL-29 and MetIL-29C172S-PEG inhibited cell death induced by BVDVin MDBK bovine kidney cells. IL-28 inhibited cell death with an IC₅₀ of0.02 μg/ml, IL-29 inhibited cell death with an IC₅₀ of 0.19 μg/ml, andMetIL-29C172S-PEG inhibited cell death with an IC₅₀ of 0.45 μg/ml. Thisdemonstrated that IL-28 and IL-29 have antiviral activity against theFlavivirida family of viruses.

Example 13 Induction of Interferon Stimulated Genes by IL-28 and IL-29A. Human Peripheral Blood Mononuclear Cells

Freshly isolated human peripheral blood mononuclear cells were grown inthe presence of IL-29 (20 ng/mL), IFN□2a (2 ng/ml) (PBL Biomedical Labs,Piscataway, N.J.), or in medium alone. Cells were incubated for 6, 24,48, or 72 hours, and then total RNA was isolated and treated withRNase-free DNase. 100 ng total RNA was used as a template for One-StepSemi-Quantitative RT-PCR® using Taqman One-Step RT-PCR Master Mix®Reagents and gene specific primers as suggested by the manufacturer.(Applied Biosystems, Branchburg, N.J.) Results were normalized to HPRTand are shown as the fold induction over the medium alone control foreach time-point. Table 15 shows that IL-29 induces Interferon StimulatedGene Expression in human peripheral blood mononuclear cells at alltime-points tested.

TABLE 15 MxA Fold Pkr Fold OAS Fold induction Induction Induction  6 hrIL29 3.1 2.1 2.5  6 hr IFNα2a 17.2 9.6 16.2 24 hr IL29 19.2 5.0 8.8 24hr IFNα2a 57.2 9.4 22.3 48 hr IL29 7.9 3.5 3.3 48 hr IFNα2a 18.1 5.017.3 72 hr IL29 9.4 3.7 9.6 72 hr IFNα2a 29.9 6.4 47.3

B. Activated Human T Cells

Human T cells were isolated by negative selection from freshly harvestedperipheral blood mononuclear cells using the Pan T-cell Isolation® kitaccording to manufacturer's instructions (Miltenyi, Auburn, Calif.). Tcells were then activated and expanded for 5 days with plate-boundanti-CD3, soluble anti-CD28 (0.5 ug/ml), (Pharmingen, San Diego, Calif.)and Interleukin 2 (IL-2; 100 U/ml) (R&D Systems, Minneapolis, Minn.),washed and then expanded for a further 5 days with IL-2. Followingactivation and expansion, cells were stimulated with IL-28A (20 ng/ml),IL-29 (20 ng/ml), or medium alone for 3, 6, or 18 hours. Total RNA wasisolated and treated with RNase-Free DNase. One-Step Semi-QuantitativeRT-PCR® was performed as described in the example above. Results werenormalized to HPRT and are shown as the fold induction over the mediumalone control for each time-point. Table 16 shows that IL-28 and IL-29induce Interferon Stimulated Gene expression in activated human T cellsat all time-points tested.

TABLE 16 MxA Fold Pkr Fold OAS Fold Induction Induction Induction Donor#1 3 hr IL28 5.2 2.8 4.8 Donor #1 3 hr IL29 5.0 3.5 6.0 Donor #1 6 hrIL28 5.5 2.2 3.0 Donor #1 6 hr IL29 6.4 2.2 3.7 Donor #1 18 hr IL28 4.64.8 4.0 Donor #1 18 hr IL29 5.0 3.8 4.1 Donor #2 3 hr IL28 5.7 2.2 3.5Donor #2 3 hr IL29 6.2 2.8 4.7 Donor #2 6 hr IL28 7.3 1.9 4.4 Donor #2 6hr IL29 8.7 2.6 4.9 Donor #2 18 hr IL28 4.7 2.3 3.6 Donor #2 18 hr IL294.9 2.1 3.8

C. Primary Human Hepatocytes

Freshly isolated human hepatocytes from two separate donors (Cambrex,Baltimore, Md. and CellzDirect, Tucson, Ariz.) were stimulated withIL-28A (50 ng/ml), IL-29 (50 ng/ml), IFNα2a (50 ng/ml), or medium alonefor 24 hours. Following stimulation, total RNA was isolated and treatedwith RNase-Free DNase. One-step semi-quantitative RT-PCR was performedas described previously in the example above. Results were normalized toHPRT and are shown as the fold induction over the medium alone controlfor each time-point. Table 17 shows that IL-28 and IL-29 induceInterferon Stimulated Gene expression in primary human hepatocytesfollowing 24-hour stimulation.

TABLE 17 MxA Fold Pkr Fold OAS Fold Induction Induction Induction Donor#1 IL28 31.4 6.4 30.4 Donor #1 IL29 31.8 5.2 27.8 Donor #1 IFN- 63.4 8.266.7 α2a Donor #2 IL28 41.7 4.2 24.3 Donor #2 IL29 44.8 5.2 25.2 Donor#2 IFN- 53.2 4.8 38.3 α2a

D. HepG2 and HuH7: Human Liver Hepatoma Cell Lines

HepG2 and HuH7 cells (ATCC NOS. 8065, Manassas, Va.) were stimulatedwith IL-28A (10 ng/ml), IL-29 (10 ng/ml), IFN□2a (10 ng/ml), IFNB (1ng/ml) (PBL Biomedical, Piscataway, N.J.), or medium alone for 24 or 48hours. In a separate culture, HepG2 cells were stimulated as describedabove with 20 ng/ml of MetIL-29C172S-PEG or MetIL-29-PEG. Total RNA wasisolated and treated with RNase-Free DNase. 100 ng Total RNA was used asa template for one-step semi-quantitative RT-PCR as describedpreviously. Results were normalized to HPRT and are shown as the foldinduction over the medium alone control for each time-point. Table 18shows that IL-28 and IL-29 induce ISG expression in HepG2 and HuH7 liverhepatoma cell lines after 24 and 48 hours.

TABLE 18 MxA Fold Pkr Fold OAS Induction Induction Fold Induction HepG224 hr IL28 12.4 0.7 3.3 HepG2 24 hr IL29 36.6 2.2 6.4 HepG2 24 hr IFNα2a12.2 1.9 3.2 HepG2 24 hr IFNβ 93.6 3.9 19.0 HepG2 48 hr IL28 2.7 0.9 1.1HepG2 48 hr IL29 27.2 2.1 5.3 HepG2 48 hr IFNα2a 2.5 0.9 1.2 HepG2 48 hrIFNβ 15.9 1.8 3.3 HuH7 24 hr IL28 132.5 5.4 52.6 HuH7 24 hr IL29 220.27.0 116.6 HuH7 24 hr IFNα2a 157.0 5.7 67.0 HuH7 24 hr IFNβ 279.8 5.6151.8 HuH7 48 hr IL28 25.6 3.4 10.3 HuH7 48 hr IL29 143.5 7.4 60.3 HuH748 hr IFNα2a 91.3 5.8 32.3 HuH7 48 hr IFNβ 65.0 4.2 35.7

TABLE 19 MxA Fold OAS Fold Pkr Fold Induction Induction InductionMetIL-29-PEG 36.7 6.9 2.2 MetIL-29C172S-PEG 46.1 8.9 2.8

Data shown is for 20 ng/ml metIL-29-PEG and metIL-29C172S-PEG versionsof IL-29 after culture for 24 hours.

Data shown is normalized to HPRT and shown as fold induction overunstimulated cells.

Example 14 Antiviral Activity of IL-28 and IL-29 in HCV Replicon System

The ability of antiviral drugs to inhibit HCV replication can be testedin vitro with the HCV replicon system. The replicon system consists ofthe Huh7 human hepatoma cell line that has been transfected withsubgenomic RNA replicons that direct constitutive replication of HCVgenomic RNAs (Blight, K. J. et al. Science 290:1972-1974, 2000).Treatment of replicon clones with IFNα at 10 IU/ml reduces the amount ofHCV RNA by 85% compared to untreated control cell lines. The ability ofIL-28A and IL-29 to reduce the amount of HCV RNA produced by thereplicon clones in 72 hours indicates the antiviral state conferred uponHuh7 cells by IL-28A/IL-29 treatment is effective in inhibiting HCVreplicon replication, and thereby, very likely effective in inhibitingHCV replication.

The ability of IL-28A and IL-29 to inhibit HCV replication as determinedby Bayer Branched chain DNA kit, is be done under the followingconditions:

IL28 alone at increasing concentrations (6)* up to 1.0 μg/ml

IL29 alone at increasing concentrations (6)* up to 1.0 μg/ml

PEGIL29 alone at increasing concentrations (6)* up to 1.0 μg/ml

IFN□2A alone at 0.3, 1.0, and 3.0 IU/ml

Ribavirin alone.

The positive control is IFNα and the negative control is ribavirin.

The cells are stained after 72 hours with Alomar Blue to assessviablility.

*The concentrations for conditions 1-3 are:

μg/ml, 0.32 μg/ml, 0.10 μg/ml, 0.032 μg/ml, 0.010 μg/ml, 0.0032 μg/ml.

The replicon clone (BB7) is treated 1× per day for 3 consecutive dayswith the doses listed above. Total HCV RNA is measured after 72 hours.

Example 15 IL-28 and IL-29 have Antiviral Activity Against PathogenicViruses

Two methods are used to assay in vitro antiviral activity of IL-28 andIL-29 against a panel of pathogenic viruses including, among others,adenovirus, parainfluenza virus. respiratory syncytial virus, rhinovirus, coxsackie virus, influenza virus, vaccinia virus, west nilevirus, dengue virus, venezuelan equine encephalitis virus, pichindevirus and polio virus. These two methods are inhibition of virus-inducedcytopathic effect (CPE) determined by visual (microscopic) examinationof the cells and increase in neutral red (NR) dye uptake into cells. Inthe CPE inhibition method, seven concentrations of test drug (log 10dilutions, such as 1000, 100, 10, 1, 0.1, 0.01, 0.001 ng/ml) areevaluated against each virus in 96-well flat-bottomed microplatescontaining host cells. The compounds are added 24 hours prior to virus,which is used at a concentration of approximately 5 to 100 cell cultureinfectious doses per well, depending upon the virus, which equates to amultiplicity of infection (MOI) of 0.01 to 0.0001 infectious particlesper cell. The tests are read after incubation at 37° C. for a specifiedtime sufficient to allow adequate viral cytopathic effect to develop. Inthe NR uptake assay, dye (0.34% concentration in medium) is added to thesame set of plates used to obtain the visual scores. After 2 h, thecolor intensity of the dye absorbed by and subsequently eluted from thecells is determined using a microplate autoreader. Antiviral activity isexpressed as the 50% effective (virus-inhibitory) concentration (EC50)determined by plotting compound concentration versus percent inhibitionon semilogarithmic graph paper. The EC50/IC50 data in some cases may bedetermined by appropriate regression analysis software. In general, theEC50s determined by NR assay are two- to fourfold higher than thoseobtained by the CPE method.

TABLE 20 Visual Assay SI Visual Virus Cell line Drug EC50 Visual IC50Visual (IC50/EC50) Adenovirus A549 IL-28A >10 μg/ml >10 μg/ml 0Adenovirus A549 IL-29 >10 μg/ml >10 μg/ml 0 Adenovirus A549MetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEG Parainfluenza MA-104 IL-28A >10μg/ml >10 μg/ml 0 virus Parainfluenza MA-104 IL-29 >10 μg/ml >10 μg/ml 0virus Parainfluenza MA-104 MetIL-29C172S- >10 μg/ml >10 μg/ml 0 virusPEG Respiratory MA-104 IL-28A >10 μg/ml >10 μg/ml 0 syncytial virusRespiratory MA-104 IL-29 >10 μg/ml >10 μg/ml 0 syncytial virusRespiratory MA-104 MetIL-29C172S- >10 μg/ml >10 μg/ml 0 syncytial PEGvirus Rhino 2 KB IL-28A >10 μg/ml >10 μg/ml 0 Rhino 2 KB IL-29 >10μg/ml >10 μg/ml 0 Rhino 2 KB MetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEGRhino 9 HeLa IL-28A >10 μg/ml >10 μg/ml 0 Rhino 9 HeLa IL-29 >10μg/ml >10 μg/ml 0 Rhino 9 HeLa MetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEGCoxsackie KB IL-28A >10 μg/ml >10 μg/ml 0 B4 virus Coxsackie KBIL-29 >10 μg/ml >10 μg/ml 0 B4 virus Coxsackie KB MetIL-29C172S- >10μg/ml >10 μg/ml 0 B4 virus PEG Influenza Maden- IL-28A >10 μg/ml >10μg/ml 0 (type A Darby [H3N2]) Canine Kidney Influenza Maden- IL-29 >10μg/ml >10 μg/ml 0 (type A Darby [H3N2]) Canine Kidney Influenza Maden-MetIL-29C172S- >10 μg/ml >10 μg/ml 0 (type A Darby PEG [H3N2]) CanineKidney Influenza Vero IL-28A 0.1 μg/ml >10 μg/ml >100 (type A [H3N2])Influenza Vero IL-29 >10 μg/ml >10 μg/ml 0 (type A [H3N2]) InfluenzaVero MetIL-29C172S- 0.045 μg/ml >10 μg/ml >222 (type A PEG [H3N2])Vaccinia Vero IL-28A >10 μg/ml >10 μg/ml 0 virus Vaccinia Vero IL-29 >10μg/ml >10 μg/ml 0 virus Vaccinia Vero MetIL-29C172S- >10 μg/ml >10 μg/ml0 virus PEG West Nile Vero IL-28A 0.00001 μg/ml >10 μg/ml >1,000,000virus West Nile Vero IL-29 0.000032 μg/ml >10 μg/ml >300,000 virus WestNile Vero MetIL-29C172S- 0.001 μg/ml >10 μg/ml >10,000 virus PEG Denguevirus Vero IL-28A 0.01 μg/ml >10 μg/ml >1000 Dengue virus Vero IL-290.032 μg/ml >10 μg/ml >312 Dengue virus Vero MetIL-29C172S- 0.0075μg/ml >10 μg/ml >1330 PEG Venezuelan Vero IL-28A 0.01 μg/ml >10μg/ml >1000 equine encephalitis virus Venezuelan Vero IL-29 0.012μg/ml >10 μg/ml >833 equine encephalitis virus Venezuelan VeroMetIL-29C172S- 0.0065 μg/ml >10 μg/ml >1538 equine PEG encephalitisvirus Pichinde BSC-1 IL-28A >10 μg/ml >10 μg/ml 0 virus Pichinde BSC-1IL-29 >10 μg/ml >10 μg/ml 0 virus Pichinde BSC-1 MetIL-29C172S- >10μg/ml >10 μg/ml 0 virus PEG Polio virus Vero IL-28A >10 μg/ml >10 μg/ml0 Polio virus Vero IL-29 >10 μg/ml >10 μg/ml 0 Polio virus VeroMetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEG

TABLE 21 Neutral Red Assay SI NR Virus Cell line Drug EC50 NR IC50 NR(IC50/EC50) Adenovirus A549 IL-28A >10 μg/ml >10 μg/ml 0 Adenovirus A549IL-29 >10 μg/ml >10 μg/ml 0 Adenovirus A549 MetIL-29C172S- >10 μg/ml >10μg/ml 0 PEG Parainfluenza MA-104 IL-28A >10 μg/ml >10 μg/ml 0 virusParainfluenza MA-104 IL-29 >10 μg/ml >10 μg/ml 0 virus ParainfluenzaMA-104 MetIL-29C172S- >10 μg/ml >10 μg/ml 0 virus PEG Respiratory MA-104IL-28A >10 μg/ml >10 μg/ml 0 syncytial virus Respiratory MA-104IL-29 >10 μg/ml >10 μg/ml 0 syncytial virus Respiratory MA-104MetIL-29C172S- 5.47 μg/ml >10 μg/ml >2 syncytial virus PEG Rhino 2 KBIL-28A >10 μg/ml >10 μg/ml 0 Rhino 2 KB IL-29 >10 μg/ml >10 μg/ml 0Rhino 2 KB MetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEG Rhino 9 HeLa IL-28A1.726 μg/ml >10 μg/ml >6 Rhino 9 HeLa IL-29 0.982 μg/ml >10 μg/ml >10Rhino 9 HeLa MetIL-29C172S- 2.051 μg/ml >10 μg/ml >5 PEG Coxsackie B4 KBIL-28A >10 μg/ml >10 μg/ml 0 virus Coxsackie B4 KB IL-29 >10 μg/ml >10μg/ml 0 virus Coxsackie B4 KB MetIL-29C172S- >10 μg/ml >10 μg/ml 0 virusPEG Influenza (type Maden- IL-28A >10 μg/ml >10 μg/ml 0 A [H3N2]) DarbyCanine Kidney Influenza (type Maden- IL-29 >10 μg/ml >10 μg/ml 0 A[H3N2]) Darby Canine Kidney Influenza (type Maden- MetIL-29C172S- >10μg/ml >10 μg/ml 0 A [H3N2]) Darby PEG Canine Kidney Influenza (type VeroIL-28A 0.25 μg/ml >10 μg/ml >40 A [H3N2]) Influenza (type Vero IL-29 2μg/ml >10 μg/ml >5 A [H3N2]) Influenza (type Vero MetIL-29C172S- 1.4μg/ml >10 μg/ml >7 A [H3N2]) PEG Vaccinia virus Vero IL-28A >10μg/ml >10 μg/ml 0 Vaccinia virus Vero IL-29 >10 μg/ml >10 μg/ml 0Vaccinia virus Vero MetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEG West Nilevirus Vero IL-28A 0.0001 μg/ml >10 μg/ml >100,000 West Nile virus VeroIL-29 0.00025 μg/ml >10 μg/ml >40,000 West Nile virus VeroMetIL-29C172S- 0.00037 μg/ml >10 μg/ml >27,000 PEG Dengue virus VeroIL-28A 0.1 μg/ml >10 μg/ml >100 Dengue virus Vero IL-29 0.05 μg/ml >10μg/ml >200 Dengue virus Vero MetIL-29C172S- 0.06 μg/ml >10 μg/ml >166PEG Venezuelan Vero IL-28A 0.035 μg/ml >10 μg/ml >286 equineencephalitis virus Venezuelan Vero IL-29 0.05 μg/ml >10 μg/ml >200equine encephalitis virus Venezuelan Vero MetIL-29C172S- 0.02 μg/ml >10μg/ml >500 equine PEG encephalitis virus Pichinde virus BSC-1 IL-28A >10μg/ml >10 μg/ml 0 Pichinde virus BSC-1 IL-29 >10 μg/ml >10 μg/ml 0Pichinde virus BSC-1 MetIL-29C172S- >10 μg/ml >10 μg/ml 0 PEG Poliovirus Vero IL-28A >1.672 μg/ml >10 μg/ml >6 Polio virus Vero IL-29 >10μg/ml >10 μg/ml 0 Polio virus Vero MetIL-29C172S- >10 μg/ml >10 μg/ml 0PEG

Example 16 IL-28, IL-29, metIL-29-PEG and metIL-29C172S-PEG StimulateISG Induction in the Mouse Liver Cell Line AML-12

Interferon stimulated genes (ISGs) are genes that are induced by type Iinterferons (IFNs) and also by the IL-28 and IL-29 family molecules,suggesting that IFN and IL-28 and IL-29 induce similar pathways leadingto antiviral activity. Human type I IFNs (IFN□1-4 and IFN□) have littleor no activity on mouse cells, which is thought to be caused by lack ofspecies cross-reactivity. To test if human IL-28 and IL-29 have effectson mouse cells, ISG induction by human IL-28 and IL-29 was evaluated byreal-time PCR on the mouse liver derived cell line AML-12.

AML-12 cells were plated in 6-well plates in complete DMEM media at aconcentration of 2×10⁶ cells/well. Twenty-four hours after platingcells, human IL-28 and IL-29 were added to the culture at aconcentration of 20 ng/ml. As a control, cells were either stimulatedwith mouse IFN□ (positive control) or unstimulated (negative). Cellswere harvested at 8, 24, 48 and 72 hours after addition of CHO-derivedhuman IL-28A (SEQ ID NO:18) or IL-29 (SEQ ID NO:20). RNA was isolatedfrom cell pellets using RNAEasy-Kit™ (Qiagen, Valencia, Calif.). RNA wastreated with DNase (Millipore, Billerica, Mass.) to clean RNA of anycontaminating DNA. cDNA was generated using Perkin-Elmer RT mix. ISGgene induction was evaluated by real-time PCR using primers and probesspecific for mouse OAS, Pkr and Mx1. To obtain quantitative data, HPRTreal-time PCR was duplexed with ISG PCR. A standard curve was obtainedusing known amounts of RNA from IFN-stimulated mouse PBLs. All data areshown as expression relative to internal HPRT expression.

Human IL-28A and IL-29 stimulated ISG induction in the mouse hepatocytecell line AML-12 and demonstrated that unlike type I IFNs, the IL-28/29family proteins showed cross-species reactivity.

TABLE 22 Stimulation OAS PkR Mx1 None 0.001 0.001 0.001 Human IL-28 0.040.02 0.06 Human IL-29 0.04 0.02 0.07 Mouse IL-28 0.04 0.02 0.08 MouseIFNα 0.02 0.02 0.01

All data shown were expressed as fold relative to HPRT gene expressionng of OAS mRNA=normalized value of OAS mRNA amount relative to internal

ng of HPRT mRNA housekeeping gene, HPRT

As an example, the data for the 48 hour time point is shown.

TABLE 23 AML12's Mx1 Fold OAS Fold Pkr Fold Induction InductionInduction MetIL-29-PEG 728 614 8 MetIL-29C172S-PEG 761 657 8

Cells were stimulated with 20 ng/ml metIL-29-PEG or metIL-29C172S-PEGfor 24 hours.

Data shown is normalized to HPRT and shown as fold induction overunstimulated cells.

Example 17 ISGs are Efficiently Induced in Spleens of Transgenic MiceExpressing Human IL-29

Transgenic (Tg) mice were generated expressing human IL-29 under thecontrol of the Eu-lck promoter. To study if human IL-29 has biologicalactivity in vivo in mice, expression of ISGs was analyzed by real-timePCR in the spleens of Eu-lck IL-29 transgenic mice.

Transgenic mice (C3H/C57BL/6) were generated using a construct thatexpressed the human IL-29 gene under the control of the Eu-lck promoter.This promoter is active in T cells and B cells. Transgenic mice andtheir non-transgenic littermates (n=2/gp) were sacrificed at about 10weeks of age. Spleens of mice were isolated. RNA was isolated from cellpellets using RNAEasy-Kit® (Qiagen). RNA was treated with DNase to cleanRNA of any contaminating DNA. cDNA was generated using Perkin-Elmer RT®mix. ISG gene induction was evaluated by real-time PCR using primers andprobes (5′ FAM, 3′ NFQ) specific for mouse OAS, Pkr and Mx1. To obtainquantitative data, HPRT real-time PCR was duplexed with ISG PCR.Furthermore, a standard curve was obtained using known amounts of IFNstimulated mouse PBLs. All data are shown as expression relative tointernal HPRT expression.

Spleens isolated from IL-29 Tg mice showed high induction of ISGs OAS,Pkr and Mx1 compared to their non-Tg littermate controls suggesting thathuman IL-29 is biologically active in vivo in mice.

TABLE 24 Mice OAS PkR Mx1 Non-Tg 4.5 4.5 3.5 IL-29 Tg 12 8 21

All data shown are fold expression relative to HPRT gene expression. Theaverage expression in two mice is shown

Example 18 Human IL-28 and IL-29 Protein Induce ISG Gene Expression inLiver, Spleen and Blood of Mice

To determine whether human IL-28 and IL-29 induce interferon stimulatedgenes in vivo, CHO-derived human IL-28A and IL-29 protein were injectedinto mice. In addition, E. coli derived IL-29 was also tested in in vivoassays as described above using MetIL-29C172S-PEG and MetIL-29-PEG. Atvarious time points and at different doses, ISG gene induction wasmeasured in the blood, spleen and livers of the mice.

C57BL/6 mice were injected i.p or i.v with a range of doses (10 μg-250μg) of CHO-derived human IL-28A and IL-29 or MetIL-29C172S-PEG andMetIL-29C16-C113-PEG. Mice were sacrificed at various time points (1hr-48 hr). Spleens and livers were isolated from mice, and RNA wasisolated. RNA was also isolated from the blood cells. The cells werepelleted and RNA isolated from pellets using RNAEasy®-kit (Qiagen). RNAwas treated with DNase (Amicon) to rid RNA of any contaminating DNA.cDNA was generated using Perkin-Elmer RT mix (Perkin-Elmer). ISG geneinduction was measured by real-time PCR using primers and probesspecific for mouse OAS, Pkr and Mx1. To obtain quantitative data, HPRTreal-time PCR was duplexed with ISG PCR. A standard curve was calculatedusing known amounts of IFN-stimulated mouse PBLs. All data are shown asexpression relative to internal HPRT expression.

Human IL-29 induced ISG gene expression (OAS, Pkr, Mx1) in the livers,spleen and blood of mice in a dose dependent manner. Expression of ISGspeaked between 1-6 hours after injection and showed sustained expressionabove control mice upto 48 hours. In this experiment, human IL-28A didnot induce ISG gene expression.

TABLE 25 Injection OAS-1 hr OAS-6 hr OAS-24 hr OAS-48 hr None - liver1.6 1.6 1.6 1.6 IL-29 liver 2.5 4 2.5 2.8 None - spleen 1.8 1.8 1.8 1.8IL-29-spleen 4 6 3.2 3.2 None - blood 5 5 5 5 IL-29 blood 12 18 11 10

Results shown are fold expression relative to HPRT gene expression. Asample data set for IL-29 induced OAS in liver at a single injection of250 μg i.v. is shown. The data shown is the average expression from 5different animals/group.

TABLE 26 Injection OAS (24 hr) None 1.8 IL-29 10 μg 3.7 IL-29 50 μg 4.2IL-29 250 μg 6

TABLE 27 MetIL-29-PEG MetIL-29C172S-PEG Naive 3 hr 6 hr 12 hr 24 hr 3 hr6 hr 12 hr 24 hr 24 hr PKR 18.24 13.93 4.99 3.77 5.29 5.65 3.79 3.553.70 OAS 91.29 65.93 54.04 20.81 13.42 13.02 10.54 8.72 6.60 Mx1 537.51124.99 33.58 35.82 27.89 29.34 16.61 0.00 10.98

Mice were injected with 100 μg of proteins i.v. Data shown is foldexpression over HPRT expression from livers of mice. Similar data wasobtained from blood and spleens of mice.

Example 19 IL-28 and IL-29 Induce ISG Protein in Mice

To analyze of the effect of human IL-28 and IL-29 on induction of ISGprotein (OAS), serum and plasma from IL-28 and IL-29 treated mice weretested for OAS activity.

C57BL/6 mice were injected i.v with PBS or a range of concentrations (10μg-250 μg) of human IL-28 or IL-29. Serum and plasma were isolated frommice at varying time points, and OAS activity was measured using the OASradioimmunoassay (RIA) kit from Eiken Chemicals (Tokyo, Japan).

IL-28 and IL-29 induced OAS activity in the serum and plasma of miceshowing that these proteins are biologically active in vivo.

TABLE 28 Injection OAS-1 hr OAS-6 hr OAS-24 hr OAS-48 hr None 80 80 8080 IL-29 80 80 180 200

OAS activity is shown at pmol/dL of plasma for a single concentration(250 μg) of human IL-29.

Example 29 IL-28 and IL-29 Inhibit Adenoviral Pathology in Mice

To test the antiviral activities of IL-28 and IL-29 against viruses thatinfect the liver, the test samples were tested in mice againstinfectious adenoviral vectors expressing an internal green fluorescentprotein (GFP) gene. When injected intravenously, these viruses primarilytarget the liver for gene expression. The adenoviruses are replicationdeficient, but cause liver damage due to inflammatory cell infiltratethat can be monitored by measurement of serum levels of liver enzymeslike AST and ALT, or by direct examination of liver pathology.

C57B1/6 mice were given once daily intraperitoneal injections of 50 μgmouse IL-28 (zcyto24) or metIL-29C172S-PEG for 3 days. Control animalswere injected with PBS. One hour following the 3^(rd) dose, mice weregiven a single bolus intravenous tail vein injection of the adenoviralvector, AdGFP (1×10⁹ plaque-forming units (pfu)). Following this, everyother day mice were given an additional dose of PBS, mouse IL-28 ormetIL-29C172S-PEG for 4 more doses (total of 7 doses). One hourfollowing the final dose of PBS, mouse IL-28 or metIL-29C172S-PEG micewere terminally bleed and sacrificed. The serum and liver tissue wereanalyzed. Serum was analyzed for AST and ALT liver enzymes. Liver wasisolated and analyzed for GFP expression and histology. For histology,liver specimens were fixed in formalin and then embedded in paraffinfollowed by H&E staining. Sections of liver that had been blinded totreat were examined with a light microscope. Changes were noted andscored on a scale designed to measure liver pathology and inflammation.

Mouse IL-28 and IL-29 inhibited adenoviral infection and gene expressionas measured by liver fluorescence. PBS-treated mice (n=8) had an averagerelative liver fluorescence of 52.4 (arbitrary units). In contrast,IL-28-treated mice (n=8) had a relative liver fluorescence of 34.5, andIL-29-treated mice (n=8) had a relative liver fluorescence of 38.9. Areduction in adenoviral infection and gene expression led to a reducedliver pathology as measured by serum ALT and AST levels and histology.PBS-treated mice (n=8) had an average serum AST of 234 U/L (units/liter)and serum ALT of 250 U/L. In contrast, IL-28-treated mice (n=8) had anaverage serum AST of 193 U/L and serum ALT of 216 U/L, and IL-29-treatedmice (n=8) had an average serum AST of 162 U/L and serum ALT of 184 U/L.In addition, the liver histology indicated that mice given either mouseIL-28 or IL-29 had lower liver and inflammation scores than thePBS-treated group. The livers from the IL-29 group also had lessproliferation of sinusoidal cells, fewer mitotic figures and fewerchanges in the hepatocytes (e.g. vacuolation, presence of multiplenuclei, hepatocyte enlargement) than in the PBS treatment group. Thesedata demonstrate that mouse IL-28 and IL-29 have antiviral propertiesagainst a liver-trophic virus.

Example 21 LCMV Models

Lymphocytic choriomeningitis virus (LCMV) infections in mice mice are anexcellent model of acture and chronic infection. These models are usedto evaluate the effect of cytokines on the antiviral immune response andthe effects IL-28 and IL-29 have viral load and the antiviral immuneresponse. The two models used are: LCMV Armstrong (acute) infection andLCMV Clone 13 (chronic) infection. (See, e.g., Wherry et al., J. Virol.77:4911-4927, 2003; Blattman et al., Nature Med. 9(5):540-547, 2003;Hoffman et al., J. Immunol. 170:1339-1353, 2003.) There are three stagesof CD8 T cell development in response to virus: 1) expansion, 2)contraction, and 3) memory (acute model). IL-28 or IL-29 is injectedduring each stage for both acute and chronic models. In the chronicmodel, IL-28 or IL-29 is injected 60 days after infection to assess theeffect of IL-28 or IL-29 on persistent viral load. For both acute andchronic models, IL-28 or IL-29 is injected, and the viral load in blood,spleen and liver is examined. Other paramenter that can be examinedinclude: tetramer staining by flow to count the number of LCMV-specificCD8+ T cells; the ability of tetramer+ cells to produce cytokines whenstimulated with their cognate LCMV antigen; and the ability ofLCMV-specific CD8+ T cells to proliferate in response to their cognateLCMV antigen. LCMV-specific T cells are phenotyped by flow cytometry toassess the cells activation and differentiation state. Also, the abilityof LCMV-specific CTL to lyse target cells bearing their cognate LCMVantigen is examined. The number and function of LCMV-specific CD4+ Tcells is also assessed.

A reduction in viral load after treatment with IL-28 or IL-29 isdetermined. A 50% reduction in viral load in any organ, especiallyliver, would be significant. For IL-28 or IL-29 treated mice, a 20%increase in the percentage of tetramer positive T cells thatproliferate, make cytokine, or display a mature phenotype relative tountreated mice would also be considered significant.

IL-28 or IL-29 injection leading to a reduction in viral load is due tomore effective control of viral infection especially in the chronicmodel where untreated the viral titers remain elevated for an extendedperiod of time. A two fold reduction in viral titer relative tountreated mice is considered significant.

Example 22 Influenza Model of Acute Viral Infection A. PreliminaryExperiment to Test Antiviral Activity

To determine the antiviral activity of IL-28 or IL-29 on acute infectionby Influenza virus, an in vivo study using influenza infected c57B1/6mice is performed using the following protocol:

Animals: 6 weeks-old female BALB/c mice (Charles River) with 148 mice,30 per group.

Groups:

Absolute control (not infected) to run in parallel for antibody titreand histopathology (2 animals per group)

Vehicle (i.p.) saline

Amantadine (positive control) 10 mg/day during 5 days (per os) starting2 hours before infection

IL-28 or IL-29 treated (5 μg, i.p. starting 2 hours after infection)

IL-28 or IL-29 (25 μg, i.p. starting 2 hours after infection)

IL-28 or IL-29 (125 μg, i.p. starting 2 hours after infection)

Day 0—Except for the absolute controls, all animals infected withInfluenza virus

For viral load (10 at LD50)

For immunology workout (LD30)

Day 0-9—daily injections of IL-28 or IL-29 (i.p.)

Body weight and general appearance recorded (3 times/week)

Day 3—sacrifice of 8 animals per group

Viral load in right lung (TCID50)

Histopathology in left lung

Blood sample for antibody titration

Day 10—sacrifice of all surviving animals collecting blood samples forantibody titration, isolating lung lymphocytes (4 pools of 3) for directCTL assay (in all 5 groups), and quantitative immunophenotyping for thefollowing markers: CD3/CD4, CD3/CD8, CD3/CD8/CD11b, CD8/CD44/CD62L,CD3/DX5, GR-1/F480, and CD19.

Study No. 2

Efficacy study of IL-28 or IL-29 in C57B1/6 mice infected withmouse-adapted virus is done using 8 weeks-old female C57B1/6 mice(Charles River).

Group 1: Vehicle (i.p.)

Group 2: Positive control: Anti-influenza neutralizing antibody (goatanti-influenza A/USSR (HIN1) (Chemicon International, Temecula, Calif.);40 μg/mouse at 2 h and 4 h post infection (10 μl intranasal)

Group 3: IL-28 or IL-29 (5 μg, i.p.)

Group 4: IL-28 or IL-29 (25 μg, i.p.)

Group 5: IL-28 or IL-29 (125 μg, i.p.)

Following-life observations and immunological workouts are prepared:

Day 0—all animals infected with Influenza virus (dose determined inexperiment 2)

Day 0-9—daily injections of IL-28 or IL-29 (i.p.)

Body weight and general appearance recorded every other day

Day 10—sacrifice of surviving animals and perform viral assay todetermine viral load in lung.

Isolation of lung lymphocytes (for direct CTL assay in the lungs usingEL-4 as targets and different E:T ratio (based on best results fromexperiments 1 and 2).

Tetramer staining: The number of CD8+ T cells binding MHC Class Itetramers containing influenza A nucleoprotein (NP) epitope are assessedusing complexes of MHC class I with viral peptides: FLU-NP₃₆₆₋₃₇₄/D^(b)(ASNENMETM), (LMCV peptide/D^(b)).

Quantitative immunophenotyping of the following: CD8, tetramer,intracellular IFN□, NK1.1, CD8, tetramer, CD62L, CD44, CD3(+ or −),NK1.1(+), intracellular IFN

CD4, CD8, NK1.1, DX5, CD3 (+ or −), NK1.1, DX5, tetramer, Single coloursamples for cytometer adjustment.

Survival Re-Challenge Study

Day 30: Survival study with mice are treated for 9 days with differentdoses of IL-28 or IL-29 or with positive anti-influenza antibodycontrol. Body weight and antibody production in individual serum samples(Total, IgG1, IgG2a, IgG2b) are measured.

Re-Challenge Study:

Day 0: Both groups will be infected with A/PR virus (1LD30).

Group 6 will not be treated.

Group 7 will be treated for 9 days with 125 μg of IL-28 or IL-29.

Day 30: Survival study

Body weight and antibody production in individual serum samples (Total,IgG1, IgG2a, IgG2b) are measured.

Day 60: Re-challenge study

Survivors in each group will be divided into 2 subgroups

Group 6A and 7A will be re-challenge with A/PR virus (1LD30)

Group 6B and 7B will be re-challenge with A/PR virus (1LD30).

Both groups will be followed up and day of sacrifice will be determined.Body weight and antibody production in individual serum samples (Total,IgG1, IgG2a, IgG2b) are measured.

Example 21 IL-28 and IL-29 have Antiviral Activity Against Hepatitis BVirus (HBV) In Vivo

A transgenic mouse model (Guidotti et al., J. Virology 69:6158-6169,1995) supports the replication of high levels of infectious HBV and hasbeen used as a chemotherapeutic model for HBV infection. Transgenic miceare treated with antiviral drugs and the levels of HBV DNA and RNA aremeasured in the transgenic mouse liver and serum following treatment.HBV protein levels can also be measured in the transgenic mouse serumfollowing treatment. This model has been used to evaluate theeffectiveness of lamivudine and IFN-α in reducing HBV viral titers.

HBV TG mice (male) are given intraperitoneal injections of 2.5, 25 or250 micrograms IL-28 or IL-29 every other day for 14 days (total of 8doses). Mice are bled for serum collection on day of treatment (day 0)and day 7. One hour following the final dose of IL-29 mice undergo aterminal bleed and are sacrificed. Serum and liver are analyzed forliver HBV DNA, liver HBV RNA, serum HBV DNA, liver HBc, serum Hbe andserum HBs.

Reduction in liver HBV DNA, liver HBV RNA, serum HBV DNA, liver HBc,serum Hbe or serum HBs in response to IL-28 or IL-29 reflects antiviralactivity of these compounds against HBV.

Example 22 IL-28 and IL-29 Inhibit Human herpesvirus-8 (HHV-8)Replication in BCBL-1 Cells

The antiviral activities of IL-28 and IL-29 were tested against HHV-8 inan in vitro infection system using a B-lymphoid cell line, BCBL-1.

In the HHV-8 assay the test compound and a ganciclovir control wereassayed at five concentrations each, diluted in a half-log series. Theendpoints were TaqMan PCR for extracellular HHV-8 DNA (IC50) and cellnumbers using CellTiter96® reagent (TC50; Promega, Madison, Wis.).Briefly, BCBL-1 cells were plated in 96-well microtiter plates. After16-24 hours the cells were washed and the medium was replaced withcomplete medium containing various concentrations of the test compoundin triplicate. Ganciclovir was the positive control, while media alonewas a negative control (virus control, VC). Three days later the culturemedium was replaced with fresh medium containing the appropriatelydiluted test compound. Six days following the initial administration ofthe test compound, the cell culture supernatant was collected, treatedwith pronase and DNAse and then used in a real-time quantitative TaqManPCR assay. The PCR-amplified HHV-8 DNA was detected in real-time bymonitoring increases in fluorescence signals that result from theexonucleolytic degradation of a quenched fluorescent probe molecule thathybridizes to the amplified HHV-8 DNA. For each PCR amplification, astandard curve was simultaneously generated using dilutions of purifiedHHV-8 DNA. Antiviral activity was calculated from the reduction in HHV-8DNA levels (IC₅₀). A novel dye uptake assay was then employed to measurecell viability which was used to calculate toxicity (TC₅₀). Thetherapeutic index (TI) is calculated as TC₅₀/IC₅₀.

IL-28 and IL-29 inhibit HHV-8 viral replication in BCBL-1 cells. IL-28Ahad an IC₅₀ of 1 μg/ml and a TC₅₀ of >10 μg/ml (TI>10). IL-29 had anIC₅₀ of 6.5 μg/ml and a TC₅₀ of >10 μg/ml (TI>1.85). MetIL-29C172S-PEGhad an IC₅₀ of 0.14 μg/ml and a TC₅₀ of >10 μg/ml (TI>100).

Example 23 IL-28 and IL-29 Antiviral Activity Against Epstein Barr Virus(EBV)

The antiviral activities of IL-28 and IL-29 are tested against EBV in anin vitro infection system in a B-lymphoid cell line, P3HR-1. In the EBVassay the test compound and a control are assayed at five concentrationseach, diluted in a half-log series. The endpoints are TaqMan PCR forextracellular EBV DNA (IC50) and cell numbers using CellTiter96® reagent(TC50; Promega). Briefly, P3HR-1 cells are plated in 96-well microtiterplates. After 16-24 hours the cells are washed and the medium isreplaced with complete medium containing various concentrations of thetest compound in triplicate. In addition to a positive control, mediaalone is added to cells as a negative control (virus control, VC). Threedays later the culture medium is replaced with fresh medium containingthe appropriately diluted test compound. Six days following the initialadministration of the test compound, the cell culture supernatant iscollected, treated with pronase and DNAse and then used in a real-timequantitative TaqMan PCR assay. The PCR-amplified EBV DNA is detected inreal-time by monitoring increases in fluorescence signals that resultfrom the exonucleolytic degradation of a quenched fluorescent probemolecule that hybridizes to the amplified EBV DNA. For each PCRamplification, a standard curve was simultaneously generated usingdilutions of purified EBV DNA. Antiviral activity is calculated from thereduction in EBV DNA levels (IC₅₀). A novel dye uptake assay was thenemployed to measure cell viability which was used to calculate toxicity(TC₅₀). The therapeutic index (TI) is calculated as TC₅₀/IC₅₀.

Example 24 IL-28 and IL-29 Antiviral Activity Against Herpes SimplexVirus-2 (HSV-2)

The antiviral activities of IL-28 and IL-29 were tested against HSV-2 inan in vitro infection system in Vero cells. The antiviral effects ofIL-28 and IL-29 were assessed in inhibition of cytopathic effect assays(CPE). The assay involves the killing of Vero cells by the cytopathicHSV-2 virus and the inhibition of cell killing by IL-28 and IL-29. TheVero cells are propagated in Dulbecco's modified essential medium (DMEM)containing phenol red with 10% horse serum, 1% glutamine and 1%penicillin-streptomycin, while the CPE inhibition assays are performedin DMEM without phenol red with 2% FBS, 1% glutamine and 1% Pen-Strep.On the day preceding the assays, cells were trypsinized (1%trypsin-EDTA), washed, counted and plated out at 10⁴ cells/well in a96-well flat-bottom BioCoat® plates (Fisher Scientific, Pittsburgh, Pa.)in a volume of 100 μl/well. The next morning, the medium was removed anda pre-titered aliquot of virus was added to the cells. The amount ofvirus used is the maximum dilution that would yield complete cellkilling (>80%) at the time of maximal CPE development. Cell viability isdetermined using a CellTiter 96® reagent (Promega) according to themanufacturer's protocol, using a Vmax plate reader (Molecular Devices,Sunnyvale, Calif.). Compounds are tested at six concentrations each,diluted in assay medium in a half-log series. Acyclovir was used as apositive control. Compounds are added at the time of viral infection.The average background and drug color-corrected data for percent CPEreduction and percent cell viability at each concentration aredetermined relative to controls and the IC₅₀ calculated relative to theTC₅₀.

IL-28A, IL-29 and MetIL-29C172S-PEG did not inhibit cell death (IC₅₀of >10 ug/ml) in this assay. There was also no antiviral activity ofIFN□ in the assay.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (e.g., GenBank aminoacid and nucleotide sequence submissions) cited herein are incorporatedby reference. The foregoing detailed description and examples have beengiven for clarity of understanding only. No unnecessary limitations areto be understood therefrom. The invention is not limited to the exactdetails shown and described, for variations obvious to one skilled inthe art will be included within the invention defined by the claims.

1. A method of treating a herpes-simplex virus infection in a mammal,the method comprising: administering to the mammal a therapeuticallyeffective amount of an isolated polypeptide comprising amino acidresidues 1-176 of SEQ ID NO:134, wherein after administration of thepolypeptide the herpes-simplex virus load is reduced.
 2. The method ofclaim 1 wherein the polypeptide is a recombinant polypeptide.
 3. Themethod of claim 1 wherein the polypeptide is conjugated to a polyalkyloxide moiety.
 4. The method of claim 3 wherein the polyalkyl oxidemoiety is polyethylene glycol.
 5. The method of claim 4 wherein thepolyethylene glycol is monomethoxy-PEG propionaldehyde.
 6. The method ofclaim 5 wherein the monomethoxy-PEG propionaldehyde has a molecularweight of about 20 Kd or 30 Kd.
 7. The method of claim 5 wherein themonomethoxy-PEG propionaldehyde is linear or branched.
 8. The method ofclaim 5 wherein the monomethoxy-PEG propionaldehyde is conjugatedN-terminally to the polypeptide.